Methods and materials for the synthesis of organic products

ABSTRACT

This invention provides biocatalysts that are recombinant yeast cells comprising recombinant expression vectors encoding heterologous lactate dehydrogenase genes for producing lactate.

[0001] This application claims priority to U.S. Provisional ApplicationSerial No. 60/252,541, filed Nov. 22, 2000.

BACKGROUND OF THE INVENTION

[0002] 1. Technical Field

[0003] The invention relates to methods and materials involved in theproduction of organic products.

[0004] 2. Background Information

[0005] Organic products such as lactic acid have many importantindustrial uses. For example, organic acids can be used to synthesizeplastic materials as well as other products. To meet the increasing needfor organic products, more efficient and cost effective productionmethods are being developed. One such method involves the use ofbacteria. Specifically, certain bacteria can produce large quantities ofparticular organic products under certain fermentation conditions. Theuse of living bacteria as factories, however, is limited by theinability of the bacteria to grow as the organic product accumulates inthe growth media. To circumvent such limitations, various productpurification techniques have been employed during product synthesis. Inaddition, the use of microorganisms other than bacteria has beenattempted. In fact, Saccharomyces cerevisiae, which is known to be acidtolerant, has been genetically modified in an attempt to produce lacticacid. Specifically, S. cerevisiae cells were modified by providing thecells with a bovine lactate dehydrogenase cDNA and disrupting endogenouspyruvate decarboxylase genes (PDC1, PDC5, and PDC6). While thesemodified S. cerevisiae cells produced some lactic acid, cell growth wassuppressed leading to the conclusion that both cell growth and lacticacid production need improvement.

SUMMARY OF THE INVENTION

[0006] The present invention relates generally to methods and materialsfor producing organic products. Specifically, the invention providesyeast cells, methods for culturing yeast cells, methods for making yeastcells, nucleic acid constructs, and methods and materials for producingvarious organic products. The invention is based on the discovery thatparticular microorganisms (e.g., bacterial and fungal microorganisms)can be genetically manipulated to have the ability, under specificculture conditions, to grow, utilize various carbon sources for growthas well as product production, and produce a desired organic product forcommercial purposes. For example, the yeast cells provided herein cangrow and produce an organic product when cultured at low pH and hightemperature. Having the ability to grow rapidly and produce an organicproduct efficiently under, for example, low pH and high temperatureconditions is particularly advantageous. Specifically, the ability of amicroorganism to tolerate low pH obviates the need to maintain a neutralpH environment, which can be difficult and expensive during large-scaleproduction processes. In addition, the methods and materials needed torecover the desired organic product from a low pH broth can be morepractical and efficient than those required to recover the same organicproduct from a broth having a more neutral pH. For example, certainorganic acid products can precipitate out of solution as the pH dropsbelow the product's pK_(a) value, making recovery quite simple. Further,the ability of a microorganism to tolerate high temperatures obviatesthe need to maintain cool temperatures -during the growth and productionphases. Clearly, reducing the need to lower the temperature in a largevolume tank of broth during large-scale production processes makes theoverall process more efficient and less expensive. Moreover, the abilityof a microorganism to tolerate both low pH and high temperature providesa convenient method for preventing contamination by other less tolerantmicroorganisms during the large-scale production processes.

[0007] It is important to note that a critical aspect relating to theability to produce a desired organic product for commercial purposes canbe the specific productivity at which that desired organic product isproduced. For example, providing a high specific productivity using themethods and materials as described herein can allow a microorganism togenerate the energy needed for cell maintenance when exposed to cultureconditions such as low pH and high temperature. This required energy canbe generated via a fermentation pathway under substantially anaerobicconditions, rather than relying on the generation of energy via therespiratory pathway. Obtaining energy via a fermentation pathway isparticularly advantageous when producing an organic product that doesnot require the respiratory pathway since essentially all of theprovided carbon source can be used to produce the desired organicproduct.

[0008] The invention also is based on the discovery that the utilizationof a carbon source by certain genetically manipulated microorganisms canbe controlled and directed predominately towards the production ofeither biomass or a desired organic product. In general terms, theinvention involves two types of culturing processes. One culturingprocess involves culturing microorganisms under specific cultureconditions, depending on the microorganism and desired outcome, thatpromote biomass production, while the other involves a different set ofculture conditions, also dependent upon the microorganism and desiredoutcome, that promotes the production of a desired organic product.Clearly, having the ability to manipulate the utilization of a carbonsource during large-scale production processes provides manufacturerswith greater flexibility and more control than is otherwise possible.

[0009] In addition, the invention is based on the discovery that certainmicroorganisms can be genetically manipulated such that most, if notall, of a carbon source is utilized for the production of either biomassor a desired organic product. Specifically, the invention provides yeastcells that are modified such that biosynthesis pathways that divert theutilization of a carbon source away from the production of biomass orthe desired organic product are inactivated. Inactivating suchbiosynthesis pathways provides microorganisms that can efficiently growand produce the desired product.

[0010] In general, the invention features a yeast cell containing anexogenous nucleic acid molecule, with the exogenous nucleic acidmolecule encoding a polypeptide having enzymatic activity within thecell. The nucleic acid can be incorporated into the genome of the cell.The enzymatic activity leads to the formation of an organic productthat, in some embodiments, is secreted from the cell. The cell furtherhas a crabtree-negative phenotype and produces the organic product. Thecell can be, for example, from the genus Kluyveromyces, Pichia,Hansenula, Candida, Trichosporon, or Yamadazyma. The organic product canbe, for example, a fermentation product, a pyruvate-derived product, anorganic acid, or a carboxylate such as lactate. In one embodiment, thepolypeptide can have lactate dehydrogenase activity. For example, theexogenous nucleic acid can encode a bacterial lactate dehydrogenase orfungal lactate dehydrogenase such as a K. lactis fungal lactatedehydrogenase.

[0011] In another embodiment, the cell contains four exogenous nucleicacid molecules, each of the four exogenous nucleic acid moleculesencoding a different polypeptide. For example, the first of the fourexogenous nucleic acid molecules can encode a first polypeptide havinglactate dehydrogenase activity, the second can encode a secondpolypeptide having CoA-transferase activity, the third can encode athird polypeptide having lactyl-CoA dehydratase activity, and the fourthcan encode a fourth polypeptide having acrylyl-CoA hydratase activity.Such a cell can produce acrylate as the carboxylate product.Alternatively, the first of the four exogenous nucleic acid moleculescan encode a first polypeptide having 2-dehydro-3-deoxy-D-pentanoatealdolase activity, the second can encode a second polypeptide havingxylonate dehydratase activity, the third can encode a third polypeptidehaving xylonolactonase activity, and the fourth can encode a fourthpolypeptide having D-xylose dehydrogenase activity. Such a cell canproduce a carbohydrate, such as D-xylose, as the organic product.

[0012] In yet another embodiment, the cell contains six exogenousnucleic acid molecules, each of the six exogenous nucleic acid moleculesencoding a different polypeptide. For example, the first of the sixexogenous nucleic acid molecules can encode a first polypeptide having2,5-dioxovalerate dehydrogenase activity, the second can encode a secondpolypeptide having 5-dehydro-4-deoxy-D-glucarate dehydrogenase activity,the third can encode a third polypeptide having glucarate dehydrataseactivity, the fourth can encode a fourth polypeptide having aldehydedehydrogenase activity, the fifth can encode a fifth polypeptide havingglucuronolactone reductase activity, and the sixth can encode a sixthpolypeptide having L-gulonolactone oxidase activity. Such a cell canproduce a vitamin, for example L-ascorbate, as the organic product.

[0013] The organic product can contain more than three carbon atoms, andcan be, for example, an amino acid.

[0014] In another embodiment, the cell is able to catabolize a pentosecarbon such as ribose, arabinose, xylose, and lyxose.

[0015] In another embodiment the cell has reduced pyruvate decarboxylaseactivity or reduced alcohol dehydrogenase activity. For example, thecell can lack all pyruvate decarboxylase activity. The reduced pyruvatedecarboxylase activity can be due to a disrupted genetic locus, wherethe locus normally has the nucleic acid sequence that encodes pyruvatedecarboxylase. Alternatively, the cell could contain an antisensemolecule, such as a ribozyme, that corresponds to an endogenous nucleicacid sequence, where the antisense molecule reduces the pyruvatedecarboxylase activity. The cell can also contain an additionalexogenous nucleic acid molecule that functions as a killer plasmid.

[0016] In another embodiment, the enzymatic activity of the polypeptideencoded by the exogenous nucleic acid leads to the formation of theorganic product in an NADH-consuming manner.

[0017] In another embodiment, the cell produces at least about 60 gramsof the organic product for every 100 grams of glucose consumed when thecell is cultured under optimal conditions for the production of theorganic product.

[0018] In another aspect, the invention features a cell, e.g., a yeastcell, containing an exogenous nucleic acid molecule, where the exogenousnucleic acid molecule encodes a polypeptide that promotes catabolism ofa pentose carbon by the cell. The polypeptide can be, for example,xylose reductase, xylitol dehydrogenase, or xylulokinase, and thepentose carbon can be, for example, ribose, arabinose, xylose, andlyxose. The cell can further catabolize a hexose carbon and can, ifdesired, simultaneously catabolize the hexose carbon and the pentosecarbon. The hexose carbon can be, for example, allose, altrose, glucose,mannose, gulose, iodose, fructose, galactose, and talose.

[0019] In another aspect, the invention features a yeast cell containingan exogenous nucleic acid molecule, where the exogenous nucleic acidmolecule encodes a polypeptide that promotes accumulation of acetyl-CoAin the cytoplasm of the cell.

[0020] The polypeptide can be a polypeptide that has citrate lyaseactivity, or can be a mitochondrial membrane polypeptide that promotesacetyl-CoA permeability across the mitochondrial membrane. The cell canhave reduced pyruvate decarboxylase activity or reduced alcoholdehydrogenase activity. Alternatively, the yeast cell can lack ethanolproduction, and can have a growth rate under culture conditions lackingethanol and acetate that is greater than the growth rate observed for acomparable yeast cell lacking ethanol production.

[0021] In yet another aspect, the invention features a yeast cell havingreduced activity of a mitochondrial polypeptide, where the cell has acrabtree-negative phenotype. Such a cell can be from, for example, thegenus Kluyveromyces, Pichia, Hansenulo, Candida, Trichosporon, orYamadazyma. The cell can completely lack the activity. The cell cancontain a disrupted locus, where the locus normally includes a nucleicacid sequence that encodes the mitochondrial polypeptide. Themitochondrial polypeptide can be a Krebs cycle enzyme. Further, the cellcan accumulate a Krebs cycle product. The cell can include an exogenousnucleic acid molecule, where the exogenous nucleic acid molecule encodesa polypeptide having enzymatic activity within the cell, with theenzymatic activity leading to formation of an organic product, such thatthe cell produces the organic product. The organic product can be, forexample, citrate, alpha-ketoglutarate, succinate, fumarate, malate, andoxaloacetate. The polypeptide can be a polypeptide that participates inthe catabolism of lactate or acetate.

[0022] In another aspect, the invention features a method for producingan organic product. The method includes providing yeast cells, where thecells include an exogenous nucleic acid molecule that encodes apolypeptide having enzymatic activity within the cells, where theenzymatic activity leads to the formation of the organic product, andwhere the cells have a crabtree-negative phenotype, and culturing thecells with culture medium such that the organic product is produced. Theyeast cells can be from within the genus Kluyveromyces, Pichia,Hansenula, Candida, Trichosporon, or Yamadazyma. The organic product canbe a fermentation product, a pyruvate-derived product, an organicproduct containing more than three carbon atoms, a carboxylate,carbohydrate, amino acid, vitamin, or lipid product. The organic productfurther can be lactate, glycerol, acrylate, xylose, ascorbate, citrate,isocitrate, alpha-ketoglutarate, succinyl-CoA, succinate, fumarate,malate, or oxaloacetate. In some embodiments, the organic product issecreted by the cells. The method can result in cells having reducedpyruvate decarboxylase activity or reduced alcohol dehydrogenaseactivity. The enzymatic activity can lead to the formation of theorganic product in an NADH-consuming manner.

[0023] Cells made by these methods can produce at least about 60 gramsof the organic product for every 100 grams of glucose consumed when theculturing step is optimal for production of the organic product. Theculture medium, which can be liquid, can include an inhibitor ofcellular respiration, such as antimycin A, cyanide, or azide. Theculturing step can include growing the cells under aerobic growthconditions followed by contacting said cells with an inhibitor ofcellular respiration.

[0024] In an alternative embodiment, the culturing step includesincubating the cells under anaerobic culture conditions. In a furtheralternative embodiment, the culturing step includes growing the cellsunder aerobic growth conditions followed by incubating the cells underanaerobic culture conditions. The culturing step can also includeculturing the cells at a temperature greater than about 35° C.

[0025] In one embodiment, the culture medium has an organic pH valueless than about 3.0, and/or an inorganic pH value less than about 3.0.In another embodiment, the medium contains a pentose carbon such asribose, arabinose, xylose, or lyxose. The medium also can include a cornfiber hydrolysate having, for example, a pH value between about 2.0 andabout 6.5.

[0026] In another aspect, the invention features a method for producingan organic product, the method including a) providing yeast cellscontaining an exogenous nucleic acid molecule encoding a polypeptidethat promotes catabolism of a pentose carbon by the cell, where the cellcontains an enzymatic activity that leads to the formation of saidorganic product, and b) culturing the cells with culture medium suchthat the organic product is produced.

[0027] In yet another aspect, the invention features a method forproducing an organic product, the method including a) providing yeastcells, where the cells include an exogenous nucleic acid moleculeencoding a polypeptide that promotes accumulation of acetyl-CoA in thecytoplasm of the cell, and where the cell contains an enzymatic activitythat leads to the formation of the organic product, and b) culturing thecells with culture medium such that the organic product is produced.

[0028] In another aspect, the invention features a method for producingan organic product, the method including a) providing yeast cells havingreduced activity of a mitochondrial enzyme, wherein reduction of theactivity leads to the accumulation of the organic product, and b)culturing said cells with culture medium such that said organic productis produced.

[0029] In another aspect, the invention features a method for culturingyeast cells having a crabtree-negative phenotype, the method includingculturing the cells with culture medium, where the culture medium has anorganic pH value less than about 3.0 and/or an inorganic pH value lessthan about 3.0. The culturing step can include culturing the cells at atemperature greater than about 35° C. The culture medium can include aninhibitor of cellular respiration. The culture medium also can include apentose carbon. In another embodiment, the culture medium can include acorn fiber hydrolysate.

[0030] In another aspect, the invention features a method for culturingyeast cells having a crabtree-negative phenotype, the method includingculturing the cells with culture medium, where the culture mediumincludes a corn fiber hydrolysate.

[0031] In another aspect, the invention features a method for culturingyeast cells having a crabtree-negative phenotype, the method includingculturing the cells with culture medium at a temperature greater thanabout 35° C., with the culture medium having an inorganic pH value lessthan about 3.0.

[0032] In another aspect, the invention features a method for culturingyeast cells having a crabtree-negative phenotype, the method includingculturing the cells with culture medium at a temperature greater thanabout 35° C., with the culture medium including a pentose carbon.

[0033] In another aspect, the invention features a method for culturingyeast cells having a crabtree-negative phenotype, the method includingculturing the cells with culture medium at a temperature greater thanabout 35° C., with the culture medium including a corn fiberhydrolysate.

[0034] In another aspect, the invention features a nucleic acidconstruct that includes a recombination sequence and a selectedsequence, with the recombination sequence corresponding to a genomicsequence of a cell having a crabtree-negative phenotype, with thegenomic sequence encoding an enzyme expressed by the cell, and with theselected sequence encoding an enzyme that leads to the formation of anorganic product within the cell. The selected sequence can be within therecombination sequence such that the selected sequence is flanked oneach end by the recombination sequence.

[0035] In another aspect, the invention features a method for making arecombinant yeast cell, including providing a yeast cell having acrabtree-negative phenotype, selecting an end product, identifying whichexogenous enzyme or enzymes need to be added to the cell to produce theend product, identifying which endogenous enzyme or enzymes whoseactivity is to be reduced in said cell to allow production of said endproduct within said cell, adding the identified exogenous enzyme orenzymes to the provided yeast cell, and reducing the activity of theidentified endogenous enzyme or enzymes in the provided yeast cell suchthat the cell produces the end product under culture conditions.

[0036] In another aspect, the invention features a corn fiberhydrolysate, the hydrolysate having a pH value between about 2.0 andabout 6.5. The hydrolysate can include glucose, xylose, and arabinose.The hydrolysate can include about 40 grams/L glucose, about 40 grams/Lxylose, and about 20 grams/L arabinose. Alternatively, the hydrolysatecan include about 38.7 grams/L-glucose, about 39.1 grams/L-xylose, about20.7 grams/L-arabinose, and about 1.6 grams/L-furfural.

[0037] In another aspect, the invention features a method for making anorganic product, including a) culturing a microorganism under cultureconditions, where the microorganism has reduced enzymatic activity; theenzymatic activity can be pyruvate decarboxylase, alcohol dehydrogenase,aldehyde dehydrogenase, or acetyl-CoA synthase activity; themicroorganism exhibits a growth rate in the absence of ethanol andacetate that is at least about 30 percent of that observed for acorresponding microorganism not having said reduced enzymatic activity,and b) changing the culture conditions to promote production of theorganic product.

[0038] In another aspect, the invention features a method for making anorganic product, including a) culturing a microorganism under cultureconditions that promote cellular respiration, where the microorganismhas reduced enzymatic activity; the enzyme activity can be pyruvatedecarboxylase, alcohol dehydrogenase, aldehyde dehydrogenase, oracetyl-CoA synthase activity, with the microorganism exhibiting a growthrate in the absence of ethanol and acetate that is at least about 30percent of that observed for a corresponding microorganism not havingsuch reduced enzymatic activity, and b) changing the culture conditionsto reduce cellular respiration, thereby promoting production of theorganic product.

[0039] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Other features and advantages of the invention will beapparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

[0040]FIG. 1 is a diagram depicting the pHES plasmid.

[0041]FIG. 2 is a diagram depicting the pSEH plasmid.

[0042]FIG. 3 is a diagram depicting the generation of pCRII plasmidscontaining either Lh-LDH or Pa-LDH.

[0043]FIG. 4 is a diagram depicting the LDII/pCRII plasmids.

[0044]FIG. 5 is a diagram depicting the generation of pHES plasmidscontaining Lh-LDH or Pa-LDH.

[0045]FIG. 6a is a diagram depicting the generation of pyruvatedecarboxylase (PDC) knockout fragment

[0046]FIG. 6b is a diagram depicting the 5.5 kbp fragment surroundingthe K marxianus 1.7 kbp PDC1.

[0047]FIG. 6c is a diagram depicting the deletion of 400 bp of the 5.5kbp PDC homologous region and the insertion of a gene for kanamycinresistance.

[0048]FIG. 6d is a diagram depicting the 4 kb region containing thekanamycin resistance gene and the surrounding 2.3 kbp of the PDC1.

[0049]FIG. 6e is a diagram depicting the 7.5 kbp K. thermotolerans PDC1and surrounding region.

[0050]FIG. 6f is a diagram depicting the deletion of 750 bp from the 1.7kbp PDC1 gene and the insertion of the kanamycin resistance gene.

[0051]FIG. 7 is a graph plotting growth (optical density; OD) versestime (hours) for Kluyveromyces marxianus cultured under low pH (pH 2.5)and high temperature (40° C.) conditions.

[0052]FIG. 8 is a graph plotting growth (OD) verses time (hours) for K.marxianus cultured with glucose, xylose, or arabinose at 30° C.

[0053]FIG. 9 is a graph plotting growth (OD) verses time (hours) for K.marxianus cultured with a corn fiber hydrolysate at 30° C.

[0054]FIG. 10 is a graph plotting growth (OD) verses time (hours) for K.marxianus cultured at 30° C. and the indicated pH.

[0055]FIG. 11 is a graph plotting growth (OD) verses time (hours) for K.marxianus cultured at 30° C. and the indicated pH in the presence of 40grams of lactic acid.

[0056]FIG. 12 shows three graphs plotting (A) biomass production; (B)glucose consumption; and (C) ethanol production of S. uvarum and K.marxianus when cultured on mineral medium with 2% glucose under aerobicconditions.

[0057]FIG. 13 shows three graphs plotting (A) biomass production; (B)glucose consumption; and (C) ethanol production of S. uvarum and K.marxianus when cultured on mineral medium with 2% glucose underanaerobic conditions.

[0058]FIG. 14 is a plasmid map of PDCI promoter vector.

[0059]FIG. 15 is

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0060] The invention provides methods and materials related to theproduction of organic products. Specifically, the invention providesyeast cells, methods for culturing yeast cells, methods for making yeastcells, nucleic acid constructs, and methods and materials for producingvarious organic products.

[0061] The yeast cells provided herein can be used to produce organicproducts. Such organic products can be used in a wide range ofapplications. For example, organic products produced by the yeast cellsdescribed herein can be used as preservatives or additives in food,pharmaceutical, or cosmetic products, and can be used to make plastic aswell as other products.

[0062] For the purpose of this invention, an organic product is anycompound containing a carbon atom. For example, carboxylates (e.g.,lactate, acrylate, citrate, isocitrate, alpha-ketoglutarate, succinate,fumarate, malate, oxaloacetate), carbohydrates (e.g., D-xylose),alditols (e.g., xylitol, arabitol, ribitol), amino acids (e.g., glycine,tryptophan, glutamate), lipids, esters, vitamins (e.g., L-ascorbate),polyols (e.g., glycerol, 1,3-propanediol, erythritol), aldehydes,alkenes, alkynes, and lactones are organic products. Thus, an organicproduct can contain one, two, three, four, five, six, seven, eight,nine, ten or more carbon atoms. In addition, organic products can have amolecular weight that is less than about 1,000 (e.g., less than about900, 800, 700, 600, 500, 400, 300, 200, or 100). For example, D-xylose(C₅H₁₀O₅) is an organic product that has a molecular weight of 150.Further, organic products can be fermentation products. The term“fermentation product” as used herein refers to any organic compoundthat is produced by a fermentation process.

[0063] In general terms, a fermentation process involves the anaerobicenzymatic conversion of organic compounds such as carbohydrates tocompounds such as ethyl alcohol, resulting in energy in the form ofadenosine triphosphate (ATP). Thus, fermentation differs from cellularrespiration in that organic products rather than molecular oxygen areused as electron acceptors. Examples of fermentation products include,without limitation, acetate, ethanol, butyrate, and lactate.

[0064] Organic products also can be pyruvate-derived products. The term“pyruvate-derived product” as used herein refers to any compound that issynthesized from pyruvate within no more than fifteen enzymatic steps.An enzymatic step is any chemical reaction or series of reactionscatalyzed by a polypeptide having enzymatic activity. The term“polypeptide having enzymatic activity” as used herein refers to anypolypeptide that catalyzes a chemical reaction of other substanceswithout itself being destroyed or altered upon completion of thereaction or reactions. Typically, an enzymatic polypeptide catalyzes theformation of one or more products from one or more substrates. Suchpolypeptides can have any type of enzymatic activity including, withoutlimitation, the enzymatic activity associated with an enzyme such asaconitase, isocitrate dehydrogenase, ketoglutarate dehydrogenase,succinate thiokinase, succinate dehydrogenase, fumarase, malatedehydrogenase, citrate synthase, 2,5-dioxovalerate dehydrogenase,5-dehydro-4-deoxy-D-glucarate dehydrogenase, glucarate dehydratase,aldehyde dehydrogenase, glucuronolactone reductase, L-gulonolactoneoxidase, 2-dehydro-3-deoxy-D-pentanoate aldolase, xylonate dehydratase,xylonolactonase, D-xylose dehydrogenase, lactate dehydrogenase,CoA-transferase, lactyl-CoA dehydratase, or acrylyl-CoA hydratase.

[0065] It is important to note that a polypeptide having a particularenzymatic activity can be a polypeptide that is eithernaturally-occurring or non-naturally-occurring. A naturally-occurringpolypeptide is any polypeptide having an amino acid sequence as found innature, including wild-type and polymorphic polypeptides. Suchnaturally-occurring polypeptides can be obtained from any speciesincluding, without limitation, mammalian, fungal, and bacterial species.A non-naturally-occurring polypeptide is any polypeptide having an aminoacid sequence that is not found in nature. Thus, anon-naturally-occurring polypeptide can be a mutated version of anaturally-occurring polypeptide, or an engineered polypeptide. Forexample, a non-naturally-occurring polypeptide having citrate synthaseactivity can be a mutated version of a naturally-occurring polypeptidehaving citrate synthase activity that retains at least some citratesynthase activity. A polypeptide can be mutated by, for example,sequence additions, deletions, and/or substitutions.

[0066] An organic product is not a pyruvate-derived product if thatproduct is synthesized from pyruvate requiring more than fifteenenzymatic steps. Examples of pyruvate-derived products include, withoutlimitation, citrate, alpha-ketoglutarate, succinate, fumarate, malate,oxaloacetate, 2-dehydro-3-deoxy-D-xylonate, D-xylonate, D-xylonolactone,D-xylose, acrylate, acetate, ethanol, butyrate, and lactate.

[0067] For purposes of this invention, carboxylate products, which canbe in a “free acid” or “salt” form, will be referred to using the saltform nomenclature. For example, lactic acid will be referred to aslactate. Thus, in this case, it will be appreciated that the term“lactate” includes lactic acid as well as lactate.

[0068] The term “nucleic acid” as used herein encompasses both RNA andDNA, including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. In addition, nucleic acid can becircular or linear. The term “exogenous” or “heterologous” as usedherein with reference to a nucleic acid molecule and a particular cellrefers to any nucleic acid molecule that does not originate from thatparticular cell as found in nature. Thus, all non-naturally-occurringnucleic acid molecules are considered to be exogenous to a cell onceintroduced into the cell. It is important to note thatnon-naturally-occurring nucleic acid molecules can contain nucleic acidsequences or fragments of nucleic acid sequences that are found innature provided the nucleic acid molecule as a whole does not exist innature. For example, a nucleic acid molecule containing a genomic DNAsequence within an expression vector is considered to be anon-naturally-occurring nucleic acid molecule, and thus is consideredlobe exogenous to a cell once introduced into the cell, since thatnucleic acid molecule as a whole (genomic DNA plus vector DNA) does notexist in nature. Thus, any vector, autonomously replicating plasmid, orvirus (e.g., retrovirus, adenovirus, or herpes virus) that as a wholedoes not exist in nature is considered to be a non-naturally-occurringnucleic acid molecule. It follows that genomic DNA fragments produced byPCR or restriction endonuclease treatment as well as cDNA's areconsidered to be non-naturally-occurring nucleic acid molecules sincethey exist as separate molecules not found in nature. It also followsthat any nucleic acid molecule containing a promoter sequence andpolypeptide-encoding sequence (e.g., cDNA or genomic DNA) in anarrangement not found in nature is considered to be anon-naturally-occurring nucleic acid molecule.

[0069] The term “endogenous” refers to genomic material that is notexogenous. Generally, endogenous genomic material develops within anorganism, tissue, or cell, and is not inserted or modified byrecombinant technology. Endogenous genomic material does include withinits scope naturally occurring variations.

[0070] It also is important to note that a nucleic acid molecule that isnaturally-occurring can be exogenous to a particular cell. For example,an entire chromosome isolated from a cell of person X would heconsidered an exogenous nucleic acid molecule with respect to a cell ofperson Y once that chromosome is introduced into Y's cell.

[0071] As used herein, the phrase “genetically modified” refers to anorganism whose genome has been modified, for example, by addition,substitution or deletion of genetic material. Methods for adding ordeleting genetic material are known and include, but are not limited to,random mutagenesis, point mutations, including insertions, deletions andsubstitutions, knock-out technology, and transformation of an organismwith a nucleic acid sequence using recombinant technology, includingboth stable and transient transformants. The yeast cells may alsocatabolize starch, either naturally or because of a geneticmodification, and may even be genetically modified to catabolizecellulosics through the addition of, for example, ftngal basedcellulases.

[0072] 1. Yeast Cells Having a Crabtree-Negative Phenotype

[0073] The invention provides a variety of genetically manipulated yeastcells that have a crabtree-negative phenotype. Such recombinant yeastcells can be used to produce organic products. For example, theinvention provides a yeast cell that has a crabtree-negative phenotype,and contains an exogenous nucleic acid molecule that encodes apolypeptide having enzymatic activity that leads to the formation of anorganic product. Such yeast cells are within the scope of the inventionprovided they produce the organic product. It is noted that the producedorganic product can be secreted from the yeast cell, eliminating theneed to disrupt the cell membrane to retrieve the organic product.Typically, the yeast cells of the invention produce the organic productwith the yield being at least about 40 grams (e.g., at least about 45,50, 55, 65, 70, 75, 80, 85, 90, or 95 grams) of organic product forevery 100 grams of glucose consumed when cultured under optimalconditions for product production. When determining the yield of organicproduct production for a particular yeast cell, any method can be used.See, e.g, Kiers et al., Yeast, 14(5):459-469 (1998). It also is notedthat the enzymatic activity of the encoded polypeptide can lead to theformation of the organic product in an NADH-consuming manner. In otherwords, the production of the organic compound can require NADH as anenergy source. The term “NAD” refers to the co-factors that act aselectron and hydrogen carriers in particular oxidation-reductionreactions, while the term “NADH” refers to the reduced form of NAD.Examples of organic products whose synthesis requires NADH include,without limitation, lactate, ethanol, acetate, and acrylate. Typically,the yeast cells within the scope of the invention catabolize a hexosecarbon such as glucose. However, such yeast cells also can catabolize apentose carbon (e.g., ribose, arabinose, xylose, and lyxose). In otherworth, a yeast cell within the scope of the invention can eithernaturally utilize a pentose carbon, or can be engineered to utilize apentose carbon. For example, a yeast cell can be given an exogenousnucleic acid molecule that encodes xylose reductase, xylitoldehydrogenase, and/or xylulokinase such that xylose can be catabolized.The yeast cells may also catabolize starch, either naturally or becauseof a genetic modification, and may even be genetically modified tocatabolize cellulosics through the addition of, for example, fungalbased cellulases. A yeast cell having a crabtree-negative phenotype isany yeast cell that does not exhibit the crabtree effect. The term“crabtree-negative” refers to both naturally occurring and geneticallymodified organisms. Briefly, the crabtree effect is defined as theinhibition of oxygen consumption by a microorganism when cultured underaerobic conditions due to the presence of a high glucose concentration(e.g., 50 grams of glucose/L). In other worth, a yeast cell having acrabtree-positive phenotype continues to ferment irrespective of oxygenavailability due to the presence of glucose, while a yeast cell having acrabtree-negative phenotype does not exhibit glucose mediated inhibitionof oxygen consumption. Examples of yeast cells typically having acrabtree-negative phenotype include, without limitation, yeast cellsfrom the following genera: Kluyveromyces, Pichia, Hansenulo, Candida,Trichosporon, and Yamadaryrna.

[0074] As described herein, the invention provides many different typesof recombinant yeast cells capable of producing a wide variety ofdifferent organic products. For example, a yeast cell can contain anexogenous nucleic acid molecule that encodes a polypeptide havinglactate dehydrogenase activity such that lactate is produced. Examplesof such a polypeptide include, without limitation, bovine lactatedehydrogenase, bacterial lactate dehydrogenase, and fungal lactatedehydrogenase (e.g., K. lactis or K. thermotolerans fungal lactatedehydrogenase). Again, polypeptides having enzymatic activity such as alactate dehydrogenase activity can be naturally-occurring ornon-naturally-occurring. It is important to note that the yeast cellsdescribed herein can contain a single copy, or multiple copies (e.g.,about 5, 10, 20, 35, 50, 75, 100 or 150 copies), of a particularexogenous nucleic acid molecule. For example, a yeast cell can containabout 50 copies of exogenous nucleic acid molecule X. It also isimportant to note that the yeast cells described herein can contain morethan one particular exogenous nucleic acid molecule. For example, ayeast cell can contain about 50 copies of exogenous nucleic acidmolecule X as well as about 75 copies of exogenous nucleic acid moleculeY. In these eases, each different nucleic acid molecule can encode adifferent polypeptide having its own unique enzymatic activity. Forexample, a yeast cell can contain four different exogenous nucleic acidmolecules such that acrylate is produced. In this example, such a yeastcell can contain a first exogenous nucleic acid molecule that encodes apolypeptide having lactate dehydrogenase activity, a second that encodesa polypeptide having CoA-transferase activity, a third that encodes apolypeptide having lactyl-CoA dehydratase activity, and a fourth thatencodes a polypeptide having acrylyl-CoA hydratase activity. In anotherexample, a yeast cell can contain four different exogenous nucleic acidmolecules such that D-xylose is produced. Specifically, such a yeastcell can contain a first exogenous nucleic acid molecule that encodes apolypeptide having 2-dehydro-3-deoxy-D-pentanoate aldolase activity, asecond that encodes a polypeptide having xylonate dehydratase activity,a third that encodes a polypeptide having xylonolactonase activity, anda fourth that encodes a polypeptide having D-xylose dehydrogenaseactivity. In yet another example, a yeast cell can contain six differentexogenous nucleic acid molecules such that the vitamin, L-ascorbate, isproduced. Specifically, such a yeast cell can contain a first exogenousnucleic acid molecules that encodes a polypeptide having2,5-dioxovalerate dehydrogenase activity, a second that encodes apolypeptide having 5-dehydro-4-deoxy-D-glucarate dehydrogenase activity,a third that encodes a polypeptide having glucarate dehydrataseactivity, a fourth that encodes a polypeptide having aldehydedehydrogenase activity, a fifth that encodes a polypeptide havingglucuronolactone reductase activity, and a sixth that encodes apolypeptide having L-gulonolactone oxidase activity.

[0075] It is important to note that enzymatic polyp peptides can be usedsuch that the desired organic product is optically pure (e.g., about 90,95, 99% pure). For example, a polypeptide having an (L)-lactatedehydrogenase activity can be used to produce (L)-lactate.

[0076] Yeast cells within the scope of the invention also can havereduced enzymatic activity such as reduced pyruvate decarboxylase and/oralcohol dehydrogenase activity. The term “reduced” as used herein withrespect to a cell and a particular enzymatic activity refers to a lowerlevel of enzymatic activity than that measured in a comparable yeastcell of the same species. Thus, a yeast cell lacking pyruvatedecarboxylase activity is considered to have reduced pyruvatedecarboxylase activity since most, if not all, comparable yeast cellshave at least some pyruvate decarboxylase activity. Such reducedenzymatic activities can be the result of lower enzyme concentration,lower specific activity of an enzyme, or combinations thereof. Manydifferent methods can be used to make a yeast cell having reducedenzymatic activity. For example, a yeast cell can be engineered to havea disrupted enzyme-encoding locus using common mutagenesis or knock-outtechnology. See, Methods in Yeast Genetics (1997 edition), Adams,Gottschling, Kaiser, and Stems, Cold Spring Harbor Press (1998).Alternatively, antisense technology can be used to reduce enzymaticactivity. For example, a yeast cell can be engineered to contain a cDNAthat encodes an antisense molecule that prevents an enzyme from beingmade. The term “antisense molecule” as used herein encompasses anynucleic acid molecule that contains sequences that correspond to thecoding strand of an endogenous polypeptide. An antisense molecule alsocan have flanking sequences (e.g., regulatory sequences). Thus,antisense molecules can be ribozymes or antisense oligonucleotides. Aribozyme can have any general structure including, without limitation,hairpin, hammerhead, or axhead structures, provided the molecule cleavesRNA.

[0077] Yeast cells having a reduced enzymatic activity can be identifiedusing any method. For example, a yeast cell having reduced pyruvatedecarboxylase activity can be easily identified using common methods.See, Ulhrich, Methods in Enzymology 18:109-115 (1970).

[0078] 2. Yeast Cells Having a Crabtree-Positive or Crabtree-NegativePhenotype

[0079] The invention also provides a variety of genetically manipulatedyeast cells that need not have a crabtree-negative phenotype, i.e., suchcells can be either crabtree-positive or crabtree-negative. Suchrecombinant yeast cells can be used to produce organic products. Forexample, the invention provides a yeast cell containing an exogenousnucleic acid molecule that encodes a polypeptide that promotescatabolism of a pentose carbon (e.g., ribose, arabinose, xylose, andlyxose) by the cell. Specifically, a yeast cell can have an exogenousnucleic acid molecule that encodes xylose reductase, xylitoldehydrogenase, and/or xylulokinase such that xylose can be metabolizedin a more efficient manner. In addition, the yeast cells capable ofcatabolizing a pentose carbon also can be capable of catabolizing ahexose carbon (e.g., allose, altrose, glucose, mannose, gulose, iodose,galactose, and talose) either sequentially or simultaneously. Forexample, a yeast cell can be engineered such that xylose and glucose arecatabolized simultaneously. It is noted that yeast cells having anincreased ability to catabolize a pentose carbon can be used to engineeryeast cells that can produce organic products from pentose carbonsources. This characteristic is particularly advantageous since pentosecarbon sources such as xylose are generally less expensive than hexosecarbon sources such as glucose. Other carbon sources that can becatabolized include, without limitation, melibiose, sucrose, fructose,raffmose, stachyose, starch (e.g., corn starch and wheat starch), andhydrolysate (e.g., corn fiber hydrolysate and other cellulosichydrolysates).

[0080] In addition, the invention provides a yeast cell containing anexogenous nucleic acid molecule that encodes a polypeptide that promotesaccumulation of acetyl-CoA in the cytoplasm of the cell. For example, ayeast cell can have an exogenous nucleic acid molecule that encodes apolypeptide having citrate lyase activity. Alternatively, a yeast cellcan have an exogenous nucleic acid molecule that encodes a mitochondrialmembrane polypeptide that promotes acetyl-CoA permeability across themitochondrial membrane. It is noted that many yeast cells lacking theability to produce ethanol cannot grow in the absence of ethanol andacetate. Typically, a yeast cell will lack the ability to produceethanol when either pyruvate decarboxylase or alcohol dehydrogenaseactivity is lacking in some manner. For example, crabtree-positive yeast(e.g., Saccharomyces) lacking pyruvate decarboxylase activity growpoorly in the absence of ethanol and acetate.

[0081] Thus, manipulation of such crabtree-positive yeast in a mannerthat reduces ethanol production in order to redirect the utilization ofpyruvate to other organic products (e.g., lactate and acrylate) resultsin poor growth characteristics when ethanol and acetate are absent,particularly since crabtree-positive yeast limit cellular respirationwhen in the presence of glucose. As described herein, yeast cells thatcan promote accumulation of cytoplasmic acetyl-CoA in some manner otherthan that which relies on cytoplasmic acetate concentration andacetyl-CoA synthase activity can grow in the absence of ethanol andacetate even when unable to produce ethanol. It is noted that yeastcells having the ability to grow in the absence of ethanol and acetatewhile lacking the ability to produce ethanol can redirect theutilization of pyruvate to produce organic products other than ethanol.

[0082] Any type of yeast can contain an exogenous nucleic acid moleculethat encodes a polypeptide that promotes accumulation of acetyl-CoA inthe cytoplasm of the cell. For example, a yeast cell having acrabtree-negative or crabtree-positive phenotype can contain anexogenous nucleic acid molecule that encodes a polypeptide that promotesaccumulation of acetyl-CoA in the cytoplasm of the cell. Typically, suchyeast cells can be identified by (1) manipulating the cell that containsthe exogenous nucleic acid molecule such that it lacks pyruvatedecarboxylase or alcohol dehydrogenase activity, (2) determining thegrowth characteristics of the cell while culturing the cell in thepresence of titrating amounts of a respiratory inhibitor (e.g.,antimycin A, cyanide, or azide), and (3) comparing those growthcharacteristics to those observed for a comparable yeast cell that doesnot contain the exogenous nucleic acid molecule, yet that also wasmanipulated to lack pyruvate decarboxylase or alcohol dehydrogenaseactivity. Yeast cells determined to have more favorable growthcharacteristics due to the presence of the exogenous nucleic acidmolecule by such a comparison are considered to contain an exogenousnucleic acid molecule that encodes a polypeptide that promotesaccumulation of acetyl-CoA in the cytoplasm of the cell.

[0083] Yeast cells containing an exogenous nucleic acid molecule thatencodes a polypeptide that promotes accumulation of acetyl-CoA in thecytoplasm of the cell also can have reduced enzymatic activity, such asreduced pyruvate decarboxylase and/or alcohol dehydrogenase activity.For example, a yeast cell can lack the ability to produce ethanol.Typically, such yeast cells have a growth rate under culture conditionslacking ethanol and acetate that is greater (e.g., about 5, 10, 20, 35,50, 75, 100, 150, 200 percent, or more) than the growth rate observedfor comparable yeast cells (i.e., yeast cells lacking the ability toproduce ethanol) that do not contain the exogenous nucleic acid, yetwere cultured under similar conditions (i.e., culture conditions lackingethanol and acetate).

[0084] The invention also provides a yeast cell having reduced activityof a polypeptide. Such yeast cells can have a crabtree-positive orcrabtree-negative phenotype. For example, a yeast cell within the scopeof the invention can have reduced activity of a plasma membranepolypeptide (e.g., a plasma membrane transporter), a cytoplasmicpolypeptide (e.g., pyruvate decarboxylase), and/or a mitochondrialpolypeptide (e.g., pyruvate dehydrogenase). The term “plasma membranetransporter” refers to polypeptides that facilitate the movement oforganic products across the plasma membrane. Examples of such apolypeptide include, without limitation, carboxylic acid transporterssuch as JEN1 in S. cerevisiae (Genbank accession number U241 55). Theterm “mitochondrial polypeptide” refers to any polypeptide thatfunctions within the mitochondria including, without limitation,pyruvate dehydrogenase, polypeptides that participate in the catabolismof lactate or acetyl-CoA (e.g., cytochrome b2 polypeptides), and Krebscycle enzymes. Krebs cycle enzymes include aconitase, isocitratedehydrogenase, ketoglutarate dehydrogenase, succinate thiokinase,succinate dehydrogenase, fumarase, malate dehydrogenase, and citratesynthase. As described herein, a yeast cell having a reduced enzymeactivity includes a yeast cell that completely lacks a particularenzymatic activity. it is important to note that the term “reduced” asused herein with respect to a yeast cell and polypeptide activity refersto a lower level of activity than that measured in a comparable yeastcell of the same species under similar conditions. Thus, a yeast celllacking a particular transport activity is considered to have reducedtransport activity if a comparable cell has at least some transportactivity. Such reduced polypeptide activities can be the result of lowerpolypeptide concentration, lower specific activity of the polypeptide,or combinations thereof. Any of various methods can be used to make ayeast cell having reduced polypeptide activity. For example, the locushaving a nucleic acid sequence that encodes a mitochondrial polypeptidecan be rendered inactive by, for example, common mutagenesis orknock-out technology.

[0085] It is noted that yeast cells having reduced activity of amitochondrial enzyme can accumulate Krebs cycle products (e.g., citrate,isocitrate, alpha-ketoglutarate, succinyl-CoA, succinate, fumarate,malate, and oxaloacetate). For example, yeast cells having reducedfumarase activity can accumulate fumarate. In addition, the yeast cellcan contain an exogenous nucleic acid molecule that encodes apolypeptide having enzymatic activity that leads to the formation of anorganic product such that the cell produces the organic product.

[0086] It is important to note that some Krebs cycle products cannotpermeate the mitochondrial membrane (e.g., alpha-ketoglutarate andsuccinyl-CoA). Thus, reducing the activity of particular Krebs cycleenzymes will result in the accumulation of certain Krebs cycle productswithin the lumen of the mitochondria. In these cases, yeast cells havinga reduced activity of a Krebs cycle enzyme can be engineered to containone or more different exogenous nucleic acid molecules, each of whichencode a polypeptide having a different enzymatic activity, such thatthe desired Krebs cycle product accumulates within the cytoplasm. Forexample, reducing the activity of ketoglutarate dehydrogenase will leadto an accumulation of alpha-ketoglutarate, which in turn will lead to anaccumulation of isocitrate. Alpha-ketoglutarate cannot permeate themitochondrial membrane, whereas isocitrate can permeate themitochondrial membrane. Thus, isocitrate can accumulate within thecytoplasm of the cell. However, yeast cells that also contain anexogenous nucleic acid molecule that encodes a polypeptide havingisocitrate dehydrogenase activity, and express that functionalpolypeptide within the cytoplasm can produce cytoplasmicalpha-ketoglutarate. Thus, reducing the activity of particular Krebscycle enzymes while providing exogenous nucleic acid molecules thatencode the same (or different) Krebs cycle enzymes that are functionalwithin the cytoplasm can lead to the production of various Krebs cycleproducts (or products derived from Krebs cycle products) within thecytoplasm.

[0087] Further, the invention provides a yeast cell having reducedactivity of an enzyme that diverts the utilization of a carbon sourceaway from the production of either biomass or the desired organicproduct. For example, enzymes within the glycerol or certain pathwayscan be disrupted and the carbon source within the culture medium isutilized predominately for the production of biomass or the desiredorganic product. Examples of glycerol pathway enzymes include, withoutlimitation, dihydroxyacetone phosphate reductase. Examples of certainpathway enzymes include, without limitation, alpha-acetolactate synthaseand alpha-acetolactate decarboxylase. Again, any method can be used toreduce the activity of an enzyme.

[0088] Moreover, any of the yeast cells provided herein can contain anexogenous nucleic acid molecule that functions as a killer plasmid. Theterm “killer plasmid” as used herein refers to a nucleic acid moleculethat provides one species of yeast with the ability to kill anotherspecies of yeast. For example, yeast cells from the genus Kluyveromycescontaining a killer plasmid can prevent the growth of yeast from thegenus Saccharomyces. Thus, yeast cells having a killer plasmid can beused to prevent contamination problems that arise during large-scaleproduction processes. In addition, any type of killer plasmid can begiven to a yeast cell. For example, a killer plasmid isolated from K.thetis can be given to a K. marxianus yeast cell. Yeast cells containinga killer plasmid can be easily identified using common methods. See,e.g., Gunge et al., J. Bacteriol. 145:382-390 (1981); Gunge and Kitada,Eur. J. Epidemiol.,4:409-414 (1988); and Wesolowski-Louvel et al.,Nonconventional yeasts in Biotechnology; Kluyveromyces lactis, ed KlausWolf, Springer Verlag, Berlin, p. 138-201 (1996).

[0089] Likewise, any of the yeast cells provided herein can contain anexogenous nucleic acid molecule that encodes a polypeptide having anATPase activity modified such that the yeast cell becomes more tolerantto low pH environments. For example, a yeast cell can he given an ATPasethat effectively maintains a low cytoplasmic proton concentration whenthe extracellular proton concentration is high. Such polypeptides can beengineered as described by Morsomme et al. (EMBOJ. 15:5513-5526 (1996)).

[0090] It is important to note that any of the recombinant yeast cellsdescribed herein can contain any combination of the described geneticmanipulations. For example, a yeast cell having a crabtree-positivephenotype can contain an exogenous nucleic acid molecule that encodes apolypeptide having citrate lyase activity as well as an exogenousnucleic acid molecule that encodes a polypeptide having enzymaticactivity that leads to the formation of an organic product.

[0091] 3 Suitable Organisms

[0092] A variety of organisms are suitable for use in accordance withthe invention. In addition to crabtree negative and crabtree positiveyeast microorganisms such as Saccharomyces Sp., including S. cerevisiaeand S. uvarum, Kluyveromyces, including K. thermotolerans, K. lactis,and K. marxianus, Pichia, Hansenula, including H. polymorpha, Candidia,Trichosporon, Yamadazyma, including Y. styptic., or Torulasporapretoriensis, organisms from a wide array of microbial species couldalso serve as hosts for lactic acid production. For example, an organismsuch as Rhizopus oryzae, a natural producer of lactic acid, could begenetically modified for acid tolerance, yield improvement, andoptically pure lactic acid. Aspergillus spp. are also known to produce avariety of organic acids, such as citric acid, and tolerate low pH.Methods for genetically modifying Aspergillus spp. to produce lacticacid are available. Moreover, fungi such as Rhizopus and Aspergillusspp. produce enzymes that enable them to degrade starch and othercarbohydrate polymers to monomer carbohydrates for use as a carbonsource.

[0093] Prokaryotes such as Escherichia coli, Zymomonas mobilis, andBacillus spp. have been or can be genetically modified for lactic acidproduction. Microorganisms that have been identified as Bacilluscoagulans are also natural producers of lactic acid that could befurther genetically modified to improve low pH lactic acid production.Additionally, extremeophile organisms from the family Archea cantolerate extremely low pH and high temperatures. Genetic modification ofselected species from this family could provide a lactic acid producingstrain.

[0094] 4. Genetic Aspects

[0095] A nucleic acid molecule encoding a polypeptide having enzymaticactivity can be identified and obtained using any method. For example,standard nucleic acid sequencing techniques and software programs thattranslate nucleic acid sequences into amino acid sequences based on thegenetic code can be used to determine whether or not a particularnucleic acid has any sequence homology with known enzymaticpolypeptides. Sequence alignment software such as MEGALIGN® (DNASTAR,Madison, Wis., 1997) can be used to compare various sequences. Inaddition, nucleic acid molecules encoding known enzymatic polypeptidescan be mutated using common molecular cloning techniques (e.g.,site-directed mutagenesis). Possible mutations include, withoutlimitation, deletions, insertions, and base substitutions, as well ascombinations of deletions, insertions, and base substitutions. Further,nucleic acid and amino acid databases (e.g., GenBank®) can be used toidentify a nucleic acid sequence that encodes a polypeptide havingenzymatic activity. Briefly, any amino acid sequence having somehomology to a polypeptide having enzymatic activity, or any nucleic acidsequence having some homology to a sequence encoding a polypeptidehaving enzymatic activity can be used as a query to search GenBank®. Theidentified polypeptides then can be analyzed to determine whether or notthey exhibit enzymatic activity.

[0096] Nucleic acid molecules that encode a polypeptide having enzymaticactivity can be identified and obtained using common molecular cloningor chemical nucleic acid synthesis procedures and techniques, includingPCR. PCR refers to a procedure or technique in which target nucleic acidis amplified in a manner similar to that described in U.S. Pat. No.4,683,195, and subsequent modifications of the procedure describedtherein. Generally, sequence information from the ends of the region ofinterest or beyond are used to design oligonucleotide primers that areidentical or similar in sequence to opposite strands of a potentialtemplate to be amplified. Using PCR, a nucleic acid sequence can beamplified from RNA or DNA. For example, a nucleic acid sequence can beisolated by PCR amplification from total cellular RNA, total genomicDNA, and cDNA as well as from bacteriophage sequences, plasmidsequences, viral sequences, and the like. When using RNA as a source oftemplate, reverse transcriptase can be used to synthesize complimentaryDNA strands.

[0097] Further, nucleic acid hybridization techniques can be used toidentify and obtain a nucleic acid molecule that encodes a polypeptidehaving enzymatic activity. Briefly, any nucleic acid molecule thatencodes a known enzymatic polypeptide, or fragment thereof can be usedas a probe to identify a similar nucleic acid molecules by hybridizationunder conditions of moderate to high stringency. Such similar nucleicacid molecules then can be isolated, sequenced, and analyzed todetermine whether the encoded polypeptide has enzymatic activity.Hybridization can be done by Southern or Northern analysis to identify aDNA or RNA sequence, respectively, which hybridizes to a probe. Theprobe can be labeled with a radioisotope such as ³²P, an enzyme,digoxygenin, or by biotinylation. The DNA or RNA to be analyzed can beelectrophoretically separated on an agarose or polyacrylamide gel,transferred to nitrocellulose, nylon, or other suitable membrane, andhybridized with the probe using standard techniques well known in theart such as those described in sections 7.39-7.52 of Sambrook et al,(1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory,Plainview, N.Y. Typically, a probe is at least about 20 nucleotides inlength. For example, a probe corresponding to a 20 nucleotide sequencethat encodes a mammalian citrate lyase can be used to identify a nucleicacid molecule that encodes a fungal polypeptide having citric lyaseactivity. In addition, probes longer or shorter than 20 nucleotides canbe used.

[0098] Any method can be used to introduce an exogenous nucleic acidmolecule into a cell. In fact, many methods for introducing nucleic acidinto yeast cells are well known to those skilled in the art. Forexample, transformation, electroporation, conjugation, and fusion ofprotoplasts are common methods for introducing nucleic acid into yeastcells. See, e.g., Ito et al., J. Bacterol. 153:163-168 (1983); Durrenset al., Curr Genet. 18:7-12 (1990); and Becker and Guarente, Methods inEnzymology 194:182-187 (1991).

[0099] It is important to note that the exogenous nucleic acid moleculecontained within a yeast cell of the invention can be maintained withinthat cell in any form. For example, exogenous nucleic acid molecules canbe integrated into the genome of the cell or maintained in an episomalstate. In other words, a cell of the invention can be a stable ortransient transformant. In addition, the yeast cells described hereincan contain a single copy, or multiple copies (e.g., about 5, 10, 20,35, 50, 75, 100 or 150 copies), of a particular exogenous nucleic acidmolecule as described above. Methods for expressing an amino acidsequence from an exogenous nucleic acid molecule are well known to thoseskilled in the art. Such methods include, without limitation,constructing a nucleic acid such that a regulatory element promotes theexpression of a nucleic acid sequence that encodes a polypeptide.Typically, regulatory elements are DNA sequences that regulate theexpression of other DNA sequences at the level of transcription. Thus,regulatory elements include, without limitation, promoters, enhancers,and the like. Moreover, methods for expressing a polypeptide from anexogenous nucleic acid molecule in yeast are well known to those skilledin the art. For example, nucleic acid constructs that are capable ofexpressing exogenous polypeptides within Kluyveromyces are well known.See, e.g., U.S. Pat. Nos. 4,859,596 and 4,943,529.

[0100] As described herein, yeast cells within the scope of theinvention contain an exogenous nucleic acid molecule that, for example,encodes a polypeptide having enzymatic activity that leads to theformation of an organic product. Methods of identifying cells thatcontain exogenous nucleic acid are well known to those skilled in theart. Such methods include, without limitation, PCR and nucleic acidhybridization techniques such as Northern and Southern analysis. In somecases, immunohistochemistry and biochemical techniques can be used todetermine if a cell contains a particular nucleic acid by detecting theexpression of the encoded enzymatic polypeptide encoded by thatparticular nucleic acid molecule. For example, an antibody havingspecificity for an encoded enzyme can be used to determine whether ornot a particular yeast cell contains that encoded enzyme. Further,biochemical techniques can be used to determine if a cell contains aparticular nucleic acid molecule encoding an enzymatic polypeptide bydetecting an organic product produced as a result of the expression ofthe enzymatic polypeptide.

[0101] For example, detection of lactate after introduction of anexogenous nucleic acid molecule that encodes a polypeptide havinglactate dehydrogenase activity into a yeast cell that does not normallyexpress such a polypeptide can indicate that that yeast cell not onlycontains the introduced exogenous nucleic acid molecule but alsoexpresses the encoded enzymatic polypeptide from that introducedexogenous nucleic acid molecule. Methods for detecting specificenzymatic activities or the presence of particular organic products arewell known to those skilled in the art. For example, the presence oflactate can be determined as described elsewhere. See, Witte et al., J.Basic Microbiol. 29:707-716 (1989).

[0102] The invention also provides a nucleic acid construct containing arecombination sequence and a selected sequence. The term “recombinationsequence” as used herein refers to any nucleic acid sequence thatcorresponds to a genomic sequence found within a cell. The recombinationsequences described herein can be used to direct recombination eventsduring the generation of knock-out organisms. In other words, arecombination sequence can be used to specifically disrupt a locuscontaining a nucleic acid sequence that encodes a particular enzyme.

[0103] The term “selected sequence” as used herein includes any nucleicacid sequence. Typically, a selected sequence encodes a polypeptidehaving enzymatic activity that leads to the formation of an organicproduct within a cell. Thus, the nucleic acid constructs of theinvention can be used to knockout an endogenous enzyme activity and addan exogenous enzyme activity in a single step. In most cases, theselected sequence is within the recombination sequence such that theselected sequence is flanked on each end by the recombination sequence.

[0104] 5. Organic Product Production and Culturing Methods

[0105] The invention provides methods for producing organic productsusing any of the yeast cells or other microbial cells provided herein.Such methods involve providing yeast cells and culturing the providedyeast cells with culture medium such that an organic product (e.g.,glycerol, acrylate, xylose, ascorbate, lactate, citrate, isocitrate,alpha-ketoglutarate, succinyl-CoA, succinate, fimarate, malate, andoxaloacetate) is produced. In general terms, the culture media and/orculture conditions can be classified into one of two categories: thosethat promote cellular respiration and/or the production of biomass andthose that reduce cellular respiration. Typically, culture media and/orculture conditions that promote cellular respiration are used insituations where rapid growth is needed, or where the organic product tobe produced cannot be produced without cellular respiration. Suchorganic products can include, without limitation, Krebs cycle products.On the other hand, culture medium and/or culture conditions that reducecellular respiration are used in situations where rapid growth is notneeded or not desired, or where the organic product to be produced canbe produced without cellular respiration. Such organic products include,without limitation, lactate, acrylate, and xylose.

[0106] As used herein, the phrase “promote cellular respiration” or“promote biomass production” when referring to a culture conditions,means that the cell culture conditions are maintained such that thecarbon source within the culture medium is predominantly metabolized byoxidative respiration or to produce biomass. As used herein, the term“biomass” refers to the dry weight of the organism. As used herein, thephrase “predominantly metabolized to produce biomass” means that atleast about 0.3 grams biomass is produced per gram carbon source (in theform of carbohydrate) consumed (e.g., at least about 0.4, 0,45, 0.5 or0.6 grams biomass). Generally, between about 0.3 to about 0.6 gramsbiomass is produced per gram carbon source. Methods for determining theamount of biomass (cell dry weight) in a culture are known and include,for example, the methods described by Postma et al, “Enzymic analysis ofthe Crabtree effect in glucose-limited chemostat cultures ofSaccharomyces cerevisiae,” Appl Environ. Microbiol 53, 468-477 (1989);and Kliers et al., “Regulation of alcoholic fermentation in batch andchemostat cultures of Kluyveromyces lactis CBS 2359,” Yeast, 14, 459-469(1998). Methods for determining the amount of carbon source consumed areknown, and include, for example HPLC methodologies.

[0107] It should be noted that the efficiency of carbon sourceutilization may depend on the carbon source and the organism. Thus,while a complex growth media that includes carbon sources other thancarbohydrate may be used, the amount of biomass produced per gram carbonsource refers only to the amount of biomass produced per gramcarbohydrate carbon source consumed.

[0108] In general, culture medium containing an inhibitor of cellularrespiration (e.g., antimycin A, cyanide, and aside) can reduce cellularrespiration, while the absence of such inhibitors can promote cellularrespiration. Likewise, anaerobic culture conditions can reduce cellularrespiration, while aerobic culture conditions can promote cellularrespiration. An aerobic condition is any condition where oxygen isintroduced or occurs naturally and serves as a substrate for therespiratory pathway. Generally, the term “aerobic” refers to a culturecondition in which the culture media is maintained under an air flow ofat least 0.1 VVM (volume air/volume liquid/minute) (e.g., greater than0.2, 0.3, 0.4, 0.5, 1.0, 1.5 or 2.0 VVM).

[0109] If a gas other than air is used then the nominal VVM is adjustedto an air equivalent based on oxygen content of the gas. Alternately,“aerobic” can be defined as a culture media that has a dissolved oxygencontent of at least 2 percent (e.g., at least 5, 10, 20, 30, 40, 50, 60,75 or 80 percent) relative to the amount present at saturated conditionswith air at atmospheric pressure. An anaerobic condition is anycondition where oxygen is purposely or naturally made essentiallyunavailable to the respiratory pathway, leading to, for example, theproduction of a reduced product such as ethanol. Generally, a conditionwhere culture medium has a dissolved oxygen (DO) content less than about2.0% (e.g., less than about 1.5, 1.0, or 0.5%, or equal to about 0%) isconsidered an anaerobic condition. Likewise, a condition having a VVM(volume air/volume liquid/minute) less than about 0.1 (e.g., less thenabout 0.05,or equal to about 0) is considered an anaerobic condition.Typically, the term “air” as used herein with respect to VVM refers toair as it exists in the atmosphere. Other culture conditions that caninfluence cellular respiration include, without limitation, pH,temperature, and the presence of particular carbon sources (e.g.,glucose). It is important to note that some culture media and/or cultureconditions that promote cellular respiration within one species of yeastcan reduce cellular respiration within another species. For example, thepresence of glucose within culture medium reduces cellular respirationin yeast cells having a crabtree-positive phenotype while having littleor no effect on cellular respiration in yeast cells having acrabtree-negative phenotype.

[0110] Directed manipulation of culture conditions during a commercialproduction can be an important step in achieving optimal levels of adesired organic product as described herein. Typically, a yeast cellwithin the scope of the invention is grown under culture conditions thatpromote cellular respiration to produce a significant cell density. Forexample, yeast cells can be placed into a culture vessel, and given anabundance of glucose and oxygen. Typically, under conditions thatpromote cellular respiration, the doubling time for the microorganismsprovided herein is less than about 10 hours (e.g., less than about 8, 5,or 3 hours). Once the cells reach a significant density, the cultureconditions can be switched to conditions that reduce cellularrespiration such that an organic product not requiring cellularrespiration is produced. For example, the yeast cells can be transferredto a culture vessel and given an abundance of glucose, but no oxygen. Inthis case, directly manipulating the culture conditions such that theyare switchedfrom aerobic to anaerobic can produce optimal levels of adesired organic product. Alternatively, in some cases, the cells can becultured solely under conditions that promote cellular respiration suchthat an organic product requiring cellular respiration is produced. Itis noted that the cell mass within the production vessel typically isgreater than about 2 g/L (e.g., greater than about 4, 6, or 8 g/L).

[0111] During culturing, the temperature can be greater than about 35°C. (e.g., greater than about 36, 37, 38, 39, 40, 41,42, 43, 44, or 45°C.). In addition, the culture medium can be liquid. The culture mediatypically contains a carbon source. Generally, the carbon sourceincludes carbohydrate containing raw materials. Typically, the nutrientmedia also contains a nitrogen source. Preferably the nitrogen sourceincludes a combination of organic and inorganic nitrogenous compounds.In one mode of operation, it may be desired to fill a large fermentationvessel with a culture medium including all of the nutrients required andall of the carbohydrate, sufficient both for biomass production and forthe production of the desired product. The vessel can be operated underconditions such that biomass production is promoted initially, forexample, by providing aerobic conditions, and then switched to anaerobicconditions for the production of the desired product in an alternatemode of operation, a smaller vessel is used for biomass production, witha high level of nutrients and sufficient carbohydrate to produce, forexample, about 100 g/L biomass. The contents of this vessel can then betransferred to a larger vessel, containing a second culture media thatcontains less nutrients, for example, only glucose as a carbon source orother carbohydrate carbon source in water. This vessel may be operateunder anaerobic conditions for the production of the desired organicproduct. Biomass growth is reduced due to the reduced level of nutrientsand the anaerobic conditions.

[0112] In a preferred embodiment, the nutrient media is kept to only therequired materials in order to simplify recovery of the desired product.Use of aerobic growth can allow a simplified media to be used, relativeto that needed if growth under anaerobic conditions was needed. Many ofthe yeast described herein can be grown, under aerobic conditions, on amedia consisting only of sugar, an inorganic nitrogen source, traceminerals, and some vitamins. Before addition of organic product to theculture medium as a result of fermentation or other processes, theculture medium generally has a pH between about 5.0 and 7.0. However, asorganic products such as organic acids are secreted into the culturemedium by the microorganism, the pH of the culture medium tends todecrease. The term “organic pH” as used herein refers to the pH of theculture 20 medium attributed to organic compounds present in the mediumsuch as carboxylates, for example, lactic acid. The term “inorganic pH”as used herein refers to the pH attributed to inorganic compounds suchas HCl and H₂SO₄. The culture medium can have an organic pH value lessthan about 3.0 (e.g., less than about 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3,2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, or 1.5), or an inorganic pH valueless than about 3.0 (e.g., less than about 2.9, 2.8, 2.7, 2.6, 2.5, 2.4,2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, or 1.5). Any carbon source canbe used during the culturing procedure. For example, medium containing apentose carbon (e.g., ribose, arabinose, xylose, and lyxose) can beused. In addition, medium containing a corn fiber hydrolysate can beused. A corn fiber hydrolysate can have a pH value between 2.0 and 6.5.Typically, a corn fiber hydrolysate contains glucose, xylose, andarabinose. For example, a corn fiber hydrolysate can contain about 40grams/L glucose, about 40 grams/L xylose, and about 20 grams/Larabinose. For large-scale production processes, the following methodscan be used. First, a large tank (e.g., a 50-, 100-, 200-, or moregallon tank) containing appropriate culture medium with, for example,hexose and/or pentose carbons is inoculated with a particularmicroorganism. After inoculation, the culture conditions can bemanipulated such that the carbon source is used predominately to producebiomass. For example, the culture medium can be manipulated to have a pHvalue of about 7.0, a temperature of about 35° C., and a dissolvedoxygen content that creates an aerobic environment throughout the tank.It is noted that the desired organic product can be produced during thisbiomass production phase. Once a sufficient biomass is reached, thebroth containing the microorganisms can be transferred to a second tank.This second tank can be any size. For example, the second tank can belarger, smaller, or the same size as the first tank. Typically, thesecond tank is larger than the first such that additional culture mediumcan be added to the broth from the first tank. In addition, the culturemedium within this second tank can be the same as, or different from,that used in the first tank. For example, the first tank can containmedium with xylose and arabinose, while the second tank contains mediumwith glucose.

[0113] Once transferred, the culture conditions within the second tankcan be manipulated such that the carbon source is used predominately toproduce organic product wherein “organic product” includes, among otherthings, pyruvate-derived products and carbon dioxide (CO,) but does notincludes biomass (i.e., cell dry weight). As used herein, the phrase“predominantly produce a “selected organic product” or a “selectedpyruvate-derived product” when referring to a culture conditions, meansthat the carbon source within the culture medium is metabolized,typically by a fermentation process (although not necessarily), to format least 0.5 grains organic product per gram carbon source consumed(e.g., at least 0.6, 0.75 or 0.8 grains organic product). Methods fordetermining the amount of organic product produced and/or carbon sourceconsumed are known and include, for example, HPLC.

[0114] As described earlier, the efficiency of carbon source utilizationmay vary depending on the substrate and organism. Thus, while a complexgrowth media which includes carbon sources other than carbohydrate(e.g., amino acids) may be used, the amount of organic product orpyruvate-derived product produced per gram carbon source refers only tothe amount of organic product or pyruvate-derived product produced pergram carbohydrate carbon source consumed. Preferably, at this stage, nomore than 0.3 grams biomass per gram carbon source is produced (e.g., nomore than 0.2, 0.1, or 0.05 grams biomass). For example, the culturemedium can be manipulated to have a dissolved oxygen content thatcreates an anaerobic environment throughout the tank, or to contain aninhibitor of cellular respiration. In addition, the culture medium canbe manipulated such that a particular pH value (e.g., an acidic,neutral, or basic pH 10 value) is maintained. Alternatively, the pH ofthe culture can be adjusted periodically without maintaining anyparticular pH value. Typically, when producing an organic acid, the pHvalue of the culture medium is maintained above at least about 1.5(e.g., at least about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,or 7.0). Further, as the microorganism catabolizes the provided carbonsources, the temperature within the tank will increase. Thus, theculture medium can be manipulated such that a particular temperature ismaintained. Alternatively, the temperature of the culture medium can headjusted periodically without maintaining any particular temperature.Typically, a temperature less than about 35° C. (e.g., less than about34, 33, 32, 31, or 30° C.) is maintained when using heat sensitivemicroorganisms, while a temperature less than about 45° C. (e.g., lessthan about 44, 43, 42, 41, 40, 39, 38, 37, 36, or 35° C.) is maintainedwhen using heat insensitive microorganisms. It is noted that biomass canbe produced during this organic product production phase. In addition,the culture conditions within the second tank can be switched from thosethat promote product production to those that promote biomassproduction, and vice versa, one or more times. For example, the cultureconditions within the second tank can be anaerobic the majority of thetime with brief pulses of dissolved oxygen such that aerobic conditionsperiodically exist.

[0115] In another method, the anaerobic culture conditions may bemodified to increase the metabolic energy of the cultured microorganism,for example, by the addition of a terminal electron acceptor. As usedherein, the term “metabolic energy” refers to the energy (in terms ofATP) derived by the organism from an energy source (such as a carbonsource). Under some conditions, the amount of metabolic energy obtainedby the organism from the metabolism of a carbon source is greater thanthe amount of energy obtained from the same carbon source underdifferent conditions.

[0116] Living cells are highly ordered and must create order withinthemselves in order to survive and grow. To maintain order within theorganism, thousands of different chemical reactions are occurring withinthe organism at any instant in time. For example, cells need energy forbiosynthetic reactions such as DNA, RNA and protein polymerizationreactions and formation of metabolic products. Cells also need energy tobring substrates into the cell, keep metabolites within the cell,maintain a proper turgor pressure and internal pH, and for motility.Because energy cannot be created or destroyed, the cell requires an.input of energy from the environment to maintain the order. Energy isgenerally supplied from the environment in the form of electromagneticradiation or chemical energy. The energy obtained from the environmentis harnessed by the cell used by one of two general biochemicalmechanisms: substrate level phosphorylation and electron transport.Generally, under anaerobic conditions, ATP (the “cellular currency’ forenergy) is produced by substrate level phosphorylation. In substratelevel phosphorylation, energy is released from chemical bonds and isstored mainly in the form of ATP. An example of substrate levelformation is the conversion of glucose to pyruvate through glycolysis:

Glucose=2 Pyruvate+2 ATP+2 H₂

[0117] Pyruvate can be then be converted into lactic acid:

Pyruvate+2 H,=Lactate

[0118] The net energy produced by the above transformation is equivalentto 2 ATP.

[0119] Pyruvate can be further processed to tricarboxylic acid (TCA)cycle and generate additional energy and hydrogen atoms:

Pyruvate—3H₂O═3CO₂+ATP+5H₂

[0120] The net reaction for glucose respiration:

Glucose+6H₂O═6CO₂—4ATP+12H,

[0121] Thus, by substrate level phosphorylation, the completerespiration of glucose to CO₂ will provide a net energy equivalent of 4ATP and 24 hydrogen atoms. In “electron transport”, theoxidation-reduction potentials of the compounds that constitute membersof an “electron transport chain” are poised such that each member can bereduced by the reduced form of the preceding member. Thus, reducingpower, as electrons, can flow through the chain of carrier molecules toa terminal electron acceptor such as oxygen (O₂), nitrate (NO3), andfumarate. Addition of a terminal electron acceptor such as oxygen,nitrate or fumarate to a culture medium can provide the microorganismwith increased metabolic energy (e.g., increased ATP production for thesame amount of carbon source consumed).

[0122] Oxygen is the most preferred terminal electron acceptor. Forexample, if oxygen is used as a terminal electron acceptor, hydrogen canbe processed through the electron transport chain and provide the cellwith an additional 1.5 ATP per hydrogen atom and 3 ATP per oxygen atom.Generally, the amount of metabolic energy can be determined by measuringthe ratio of the amount of oxygen consumed to the amount of glucoseconsumed. Table 1 presents expected maximum and minimum improvements ofenergy yield (moles ATP per moles glucose) when oxygen is added duringproduction as a function of the product yield which is decreasing due toloss of pyruvate to TCA cycle (and, consequently to respiration).Maximum % improvement was calculated assuming a P/O ratio of 3 whereasminimum % improvement assumed a P/O ratio of 0.5. Table 2 shows theestimated maximum amount oxygen consumed per mole glucose consumed.Addition of oxygen can promote minimal growth that will sequester carbonto biosynthesis leaving a small amount of carbon available forrespiration (and, therefore, oxygen utilization). TABLE 1 Maximum %Minimum % Product Yield improvement in energy improvement in energy(g-lactate/g-glucose) yield yield 1.0   0%  0% 0.9  160%  35% 0.8  320% 70% 0.7  480% 105% 0.6  640% 140% 0.5  800% 175% 0.4  960% 210% 0.31120% 245% 0.2 1280% 280% 0.1 1440% 315% 0.0 1600% 350%

[0123] TABLE 2 Product Yield mole oxygen per (g-lactate/g-glucose) moleglucose 1.0 0.0 0.9 0.6 0.8 1.1 0.7 1.6 0.6 2.1 0.5 2.6 0.4 3.1 0.3 3.60.2 4.1 0.1 4.6 0.0 5.1

[0124] Thus, to improve the metabolic energy of the microorganisms inthe cell culture, oxygen can be added to the cell culture as a terminalelectron acceptor. Whereas the maximum molar yield of lactic acid fromglucose is 2 mole lactate per mole glucose and the molar yield of ATPfrom glucose is 2 mole ATP per mole glucose, addition of oxygen as aterminal electron acceptor allows some of the pyruvate to be channeledto the citric acid (TCA) cycle where it is converted to CO₂ and energy.Thus, supplying a terminal electron acceptor “increases the metabolicenergy’ of the microorganism.

[0125] Diverting pyruvate to the TCA cycle will tend to reduce theamount of other pyruvate-derived products (such as lactic acid)produced. For example, a 10% reduction in yield may result in thegeneration of 2.6 times more metabolic energy for the microorganism, a20% reduction in yield may result in the generation of 4.2-5 times moremetabolic energy for the microorganism, and a 50% reduction in yield mayresult in the generation of 9 times more metabolic energy for themicroorganism.

[0126] It is anticipated that in the later stages of a process, whenhigh levels of metabolic products such as lactic acid are present, thatthe cell may require more metabolic energy to maintain function.

[0127] Thus, it may be desirable to expose the microorganisms within ananaerobic culture medium to brief pulses of dissolved oxygen.Preferably, the brief pulse of dissolved oxygen' results in the culturemedium having a dissolved oxygen concentration of no greater than 0.5percent, preferably between about 0.1 and 0.5 percent. Alternately, thegrowth rate or cellular maintenance of the microorganisms duringanaerobic fermentation can be increased by the addition of otherterminal electron acceptors such as nitrate or fumarate. The oxygen isadded at a level just sufficient to increase the metabolic energy of themicroorganism while maintaining productivity at a desired level. Caremust be used to avoid excessive yield loss. This technique may also beused to help consume residual sugars and thereby to further simplifyrecovery processes.

[0128] 6. Organic Product Purification Methods

[0129] Once produced, any method can be used to isolate the desiredproduct. For example, common separation techniques can be used to removethe biomass from the broth, and common isolation procedures (e.g.,extraction, distillation, and ion-exchange procedures) can be used toobtain the organic product from the microorganism-free broth. See, U.S.Pat. No. 4,275,234; U.S. Pat. No. 5,510, 526; U.S. Pat. No. 5,831,122;U.S. Pat. No. 5,641,406; and International Patent Application Number WO93/00440. In addition, the desired organic product can be isolated whileit is being produced, or it can be isolated from the broth after theproduct production phase has been terminated. It is important to notethat the culture conditions within the second tank can be manipulatedsuch that the isolation process is improved. For example, the pH andtemperature within the second tank can be manipulated such that thedesired organic product precipitates out of solution, or is in a formmore amenable to isolation. Specifically, the pH value of organic acidscan precipitate out of solution when the pH of the broth is less thanthe pKa value for the organic acid. For example, the culture conditionswhile producing glutamic acid can be such that the pH is less than 2.19,which is the pKa value for glutamic acid. Thus, manipulating the pH,temperature, and content of the broth can facilitate organic productisolation. In addition, particular genetically manipulated yeast can beselected and/or specific culture conditions can be manipulated such thatany byproducts within the broth are such that they do not interfere withthe recovery of the desired organic product.

[0130] It will be appreciated that the methods and materials describedherein can be adapted and used in any type of culturing processincluding, without limitation, the processes commonly referred to as“continuous fermentation” and “batch fermentation” processes. Inaddition, the microorganisms used during one production process can berecovered and reused in subsequent production processes. For example,the microorganisms can be reused multiple times to produce a desiredorganic product. Further, any carbon source can be used. For example,allose, altrose, glucose, mannose, gulose, iodose, galactose, talose,melibiose, sucrose, fructose, raffinose, stachyose, ribose, arabinose,xylose, lyxose, starches such as corn starch and wheat starch, andhydrolysates such as corn fiber hydrolysates and other cellulosichydrolysates can be used as a carbon source for the production of eitherbiomass or the desired organic product. Moreover, any medium can beused. For example, standard culture media (e.g., yeast minimal mediumand YP medium (yeast extract 10 g/L, peptone broth 20 g/L)) as well asmedia such as corn steep water and corn steep liquor can be used. Asignificant advantage of the present invention is that the preferredmicroorganisms, especially when grow

[0131] In under aerobic conditions, can utilize minimal media. Theanaerobic production typically will not require additional nutrients, sothe final product can be isolated from a relatively clean fermentationbroth using any of a variety of separation techniques. Liquid-liquidextraction is a well-known technique for the separation of organic acidsfrom fermentation broths, and results in considerable purification. Withthe present invention it is believed that simpler, less costly, lessenergy-consuming systems may also be useful.

[0132] In one embodiment, the present invention uses geneticallymodified yeast having a crabtree-negative phenotype in a train-typeprocess that induces a “switch” in the metabolic pathway after acritical cell density has been reached and at which time it is desiredto dramatically increase the specific productivity of the desiredorganic product. A typical method for inducing the metabolic pathwayswitch is by moving the biomass from a highly aerated vessel to asubstantially anaerobic vessel, causing oxygen starvation. It is notedthat a common carbohydrate (e.g., glucose or xylose) can be used as thecarbon source during both the growth phase and the production phase. Theuse of a genetically modified yeast cell having a crabtree-negativephenotype can be critical to the success of this embodiment. Inaddition, the specific productivity of the desired organic product canbe critical to success. The term “specific productivity” as used hereinreflects the amount of product produced and is represented as the numberof grams of organic product produced per gram of biomass (dry weight)per hour, i.e., g/(g * hour). Typically, the specific productivity fororganic products such as lactate and acrylate is greater than about 0.1g/(g * hour), for example, greater than about 0.2 g/(g * hour), orgreater than about 0.5 g/(g * hour). By providing a high specificproductivity as described herein, the energy required for cellmaintenance may be obtained via the fermentative product pathway undersubstantially anaerobic conditions, rather than relying on aeration togenerate high amounts of energy via the respiratory pathway.

[0133] It is noted that substantially anaerobic vessels are aerated at arate of less than about 0.1 VVM. Under certain production situations, noaeration will be used. In addition, the yield (i. e., g organicproduct/g carbon source consumed) in this embodiment typically isgreater than about 70 wt %, and is produced without the addition ofcarbon sources such as ethanol and acetate. In some cases, in order toachieve the specific productivity required to generate the requiredenergy for cell maintenance, it may be necessary to enhance the pathwayfrom glucose to pyruvate in addition to providing the necessary enzymesto produce the desired product.

[0134] In another embodiment, the train-type process can be designedsuch that only the highly aerated growth vessel is equipped withsterilization capability. The anaerobic production vessel is typicallyoperated at temperatures greater than about 35° C. (e.g., greater thanabout 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45° C.). Few wild-typeyeast will be able to survive and compete with the genetically modifiedyeast at such temperatures as the pH drops during product production,especially since they will not have an enhanced fermentation pathwaythat can generate energy for cell maintenance, in addition, the yeastcan be engineered to contain “killer plasmids” as described herein,which can prevent yeast from other species from surviving. The inventionalso provides various methods for culturing yeast cells. For example, ayeast cell having a crabtree-negative phenotype can be cultured withculture medium either having an organic ph value less than about 3.0, orcontaining a corn fiber hydrolysate. Other methods for culturing yeastcells include, without limitation, culturing yeast cells having acrabtree-negative phenotype at a temperature greater than about 35° C.with culture medium either having an inorganic pH value less than about3.0, or containing a pentose carbon or corn fiber hydrolysate.

[0135] Further, the invention provides a process for making an organicproduct. This process includes growing a microorganism under cultureconditions, and changing the culture conditions to promote production ofthe organic product. In this process, the microorganism has reducedpyruvate decarboxylase, alcohol dehydrogenase, aldehyde dehydrogenase,and/or acetyl-CoA synthase activity, and exhibits a growth rate in theabsence of ethanol and acetate that is at least about 30 percent (e.g.,about 35, 40, 50, 75, 100, 150, 200 percent, or more) of that observedin a corresponding microorganism not having reduced pyruvatedecarboxylase, alcohol dehydrogenase, aldehyde dehydrogenase, and/oracetyl-CoA synthase activity. Typically, culture conditions that promotecellular respiration are used in situations where rapid growth isneeded, or where the organic product to be produced cannot be producedwithout cellular respiration, while culture conditions that reducecellular respiration are used in situations where rapid growth is notneeded, or where the organic product to be produced can be producedwithout cellular respiration.

[0136] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1 Recombinant Plasmid pHES/pSEH

[0137] 0.5 ug of plasmid pGAD424 described by Chien et al. (Proc. NatlAcad. Sci., 88:9578-9582 (1991)) was digested with the restrictionenzyme HindIII. The digested mixture was separated by gelelectrophoresis on a 0.8% agarose gel using TBE buffer. A 5.9 kbpfragment was then purified from the gel as described in Sambrook et al.,(ibid.). A complementary pair of 92 bp synthetic oligomers with multiplerestriction enzyme recognition sites was designed. The first wasdesignated fwd HES oligo and has the following sequence:5′-CCCAAGCTTGAATTCCCCGGGGGATCCCTGCAGGGTACCACGCGTAGATCTACTAGTGCGGCCGCCTCGAGTCTAGAGGGCCCAAGCTTGGG-3′ (SEQ ID NO: 1). Thesecond was designated camp hes oligo and has the following sequence:5′-CCAAGCTTGGGCCCTCTAGACTCGAGGCGGCCGCACTAGTAGATCTACGCGTGGTACCCTGCAGGGATCCCCCGGGGAATTCAAGCTTGGG-3′ (SEQ ID NO:2). 500 nmolesof the two complementary oligomers were annealed to each other byboiling for ten minutes and cooling gradually to room temperature. Thedouble stranded 92 bp DNA was digested with HindIII and ligated to theHindIII digested 5.9 kbp pGAD424. The ligation mixture was used totransform E. coli DH10B (electromax cells, Life Technologies, Rockville,Md.) by electroporation as described in Sambrook et al. (ibid.).Recombinant E. coli was plated on Luria-Bertani broth plates, and cellscontaining plasmid were selected using 100 μg/mL of the antibioticampicillin. The plasmid DNA from ampicillin resistant E. coli cloneswere screened to obtain the two plasmids pHES and pSEH (FIGS. 1 and 2).The two plasmids differ in the orientation of the synthetic oligomerwith respect to the alcohol dehydrogenase—ADHI promoter on the vector.

Example 2 PCR Amplification of Nucleic Acid Encoding LactateDehydrogenase from Lactobacillus helveticus and Pediococcus acidilactici

[0138] Genomic DNA was isolated from overnight cultures of Lactobacillushelveticus (ATCC 10797) and Pediococcus acidilactici (ATCC 25741) usingPUREGENE® genomic DNA isolation kit (Gentra systems, Minneapolis,Minn.). PCR primers were designed to isolate lactatedehydrogenase-encoding nucleic acid from L. helveticus (lh-ldh oligos)and P. acidilactici (pa-ldh oligos) genomic DNA. These primers weredesigned based on the available gene sequences for lactatedehydrogenases in the Genbank databases, and have the followingsequences: 5′ lh-ldh, 5′-CCGGGATCCATGGCAAGAGAGGAAAAACCTC-3′ (SEQ IDNO:3); 3′ lh-ldh, 5-CCAAGATCTTTATTGACGAACCTTAACGCCAG-3′ (SEQ ID NO:4);5′ pa-ldh: 5′-CCGGGATCCATGTCTAATATTCAAAATCATCAAAAAG-3′ (SEQ ID NO:5);and 3′ pa-ldh, 5′-CCAAGATCTTTATTTGTCTTGTTTTTCAGCAAG-3′ (SEQ ID NO:6).The primers were optimized using Primer Designer software obtained fromSci-ed software (Durham, N.C.). One umole of the genomic DNA was usedalong with 100 nmoles of primers. Pfu DNA polymerase (New EnglandBiolabs) was used to PCR amplify lactate dehydrogenase (LDH) nucleicacid as described in Sambrook et al. (ibid.).

[0139] A similar strategy is employed to isolate L-lactatedehydrogenase-encoding nucleic acid from genomic DNA from microorganismssuch as Bacillus sp., for example, Bacillus megaterium (ATCC 6458) orRhizopus oryzae (ATCC 76275) or any other lactate producing organism(including microorganisms such as fungi and bacteria and multicellularorganisms such mammals) or from tissue from a lactate producingorganism. Genomic DNA is isolated from a growing culture of the organismusing PUREGENE® genomic DNA isolation kit (Gentra systems, Minneapolis,Minn.). Suitable PCR primers are designed to isolate the lactatedehydrogenase-encoding nucleic acid based on the LDH gene sequences forthese species available from Genbank. Generally, one μmole of thegenomic DNA is used along with 100 nmoles of the appropriate primers.Pfu DNA polymerase (New England Biolabs) or any other suitable DNApolymerase is used to amplify lactate dehydrogenase (LDH) nucleic acidfrom the respective genomic DNA using PCR technology, for example, asdescribed in Sambrook et al. (ibid.).

[0140] Alternately, lactate dehydrogenase-encoding nucleic acid isisolated from Kluyveromyces thermotolerans (ATCC 52709), Trichodennareesci (ATCC 13631), Torulaspora pretoriensis (ATCC 36245), or any otherlactate dehydrogenase producing organism using the any of the followingmethodologies.

[0141] 1) A genomic cDNA library from one of these organisms is clonedinto an standard E. coli expression vector such as pUC19 using standardtechniques (Sambrook et at. (ibid.). An .E. coli (ldh pfi) mutant strainNZNI 11 (Bunch et al., (1997) “The ldhA gene encoding the fermentativelactate dehydrogenase of Escherichia coli,” Microbiology, 143:187-95) istransformed with this library and the cells are grown under anaerobicconditions in M9 medium supplemented with casamino acid. Any E. coli.that grows under these conditions encodes either a lactate dehydrogenaseor is a revertant in ldh or pfl. Positives (colonies that form under theanaerobic growth conditions) are screened for LDH activity using acalorimetric assay of lactic-acid specific soft-agar overlay (LASSO)that is capable of differentiating between (L)-LDH and (D)-LDH (Witte etal., 1989, Basic Microbiol. 29:707-716 (1989)). Plasmid DNA from clonessuspected of expressing 1-lactate dehydrogenase are then isolated andsequenced.

[0142] 2) K. thermotolerans ATCC 52709, T reesei ATCC 13631 andTorulaspora pretoriensis ATCC 36245 are all eukaryotes that produceL-lactic acid when cultured under anaerobic conditions (Wine et al.(Basic Microbiol. 29:707-716 (1989)), Thus, according to this method, atleast one of these strains is grown under anaerobic conditions to inducelactate dehydrogenase enzyme activity. cell free extracts is thenobtained using standard methods and subjected to known proteinpurification strategies to isolate the lactate dehydrogenase enzyme.Methods for purifying lactate dehydrogenase are known (Kelly et al.,(1978), , Affinity chromatography of bacterial lactate dehydrogenases,”Biochem J., 171:543-7). After the protein is purified, it is partiallycleaved and sequenced to determine the amino acid sequence. This aminoacid sequence is then used to design degenerate primers to isolate thegene encoding lactate dehydrogenase from the genomic DNA.

[0143] An eukaryotic LDH, such as the one isolated from K.thermotolerans or Trichoderma reesei or Torulaspora pretoriensis, mayfunction better (in terms of 15 transcriptional efficiency,translational efficiency and/or protein activity) in the yeast K.marxianus compared to an LDH from bacterial sources such as Bacillus orLactobacillus.

[0144] 3) Using the known eukaryotic lactate dehydrogenase genesequences available from Genbank, degenerate primers are designed toisolate the gene for lactate dehydrogenase from genomic DNA of K.thermotolerans ATCC 52709, T. reesei ATCC 13631 or Torulosporapretoriensis ATCC 36245. The conserved NAD+ binding site and pyruvatebinding site among LDH gene sequences is used to design degenerateprimers. One μmole of genomic DNA is used along with 100 nmoles ofprimers. Pfu DNA polymerase (New England Biolabs), or any other suitableDNA polymerase, is used to amplify fragments of the L-lactatedehydrogenase (LDH) nucleic acid according to known PCR methods, forexample, those described in Sambrook et al. (ibid.).

Example 3 Cloning of L. helveticus and P. acidilactici LDH Genes intopCRII Vector

[0145] PCR amplified LDH DNA products were ligated with pCRII vectors(FIGS. 3 and 4) using the TA cloning kit obtained from Invitrogen(Carlsbad, Calif.). The ligation mixture was then used to transform E.coli DH1OB using methods described in Sambrook et al. (ibid.). The pCRIIvectors supplied with the kit allowed for quick cloning of the PCRproducts according the manufacture's instructions. The pCRII vectorswith the LDH genes from L. helveticus and P. acidilactici are depictedin FIG. 4.

Example 4 Recombinant Plasmid pLh ldh-HES/pPa ldh-HES Having L.helveticus and P. acidilactici LDH Genes in pHES Vector

[0146] The pCRII vectors containing LDH gene from L. helveticus and P.acidilactici were digested with the appropriate restrictionendonucleases. The pHES vector was similarly digested with the samerestriction endonucleases. A 1 kbp insert containing LDH from pCRIIvectors was then ligated to the 6.0 kbp pHES vector using T4 DNA ligaseas described in Sambrook et al. (ibid.). The ligation mixture was usedto transform E. coli DH1OB (electromax cells, Life Technologies,Rockville, Md.), and recombinant clones were selected for ampicillinresistance. DNA isolated from recombinant clones was analyzed to confirmthe pLh ldh-HES and pPa ldh-HES vectors (FIG. 5). These vectors containthe genes encoding LDH from L. helvecticus and P. acidilactici in thepHES vector under the control of the yeast alcohol dehydrogenasepromoter (ADH1).

Example 5 Cloning of Bacillus sp., Rhizopus oryzae, K thermotolerans,Trichoderma reesei or Torulaspora pretoriensis LDH Gene for ExpressionUsing the Saccharomyces PDC1 Gene Promoter

[0147] Although it is possible to use the lactate dehydrogenase promoterfound in Rhizopus oryzae, K thermotolerans, Trichoderma reesei orTorulaspora pretoriensis to control expression of a lactatedehydrogenase gene cloned in K. marxianus, the PDC1 promoter fromSaccharomyces cerevisiae may be used to control expression of theisolated lactate dehydrogenase gene. Saccharomyces cerevisiae glycolyticpromoters have been successfully used to express genes in Kluyveromycesstrains. (Gellissen and Hollenberg, (1997) “Application of yeasts ingene expression studies: a comparison of Saccharomyces cerevisiae,Hansenula polymorpha and Kluyveromyces lactis—a review,” Gene,190:87-97).

[0148] Accordingly, the PDC1 promoter from Saccharomyces cerevisiae isobtained by designing suitable oligomeric primers using theSaccharomyces cerevisiae genome sequence, available in Genbank. The PDC1gene sequence and 1 Kb regions surrounding the PDC1 gene are amplifiedby PCR technologies. The resulting 4 Kb DNA fragment contains both thepromoter and terminators that control PDCI gene expression. Multiplerestriction enzyme sites are included between the promoter andterminators that control the PDCI gene expression such that a variety ofLDH genes can be inserted under the control of the PDCI promoter andterminators. The 4 Kb DNA fragment is inserted into a suitable vector,such as a pUC 19 based Saccharomyces cerevisiae or E. coli shuttlevector. The LDH gene is then introduced into the vector at one of themultiple cloning sites under the control of the promoter and terminatorssuch that lactate dehydrogenase gene expression is controlled by thePDC1 promoter and terminator. (FIG. 14).

[0149] Alternately, other Saccharomyces glycolytic promoters such asthose that control the expression of the Saccharomyces cerevisiaeglyceraldehyde-3 phosphate dehydrogenase or the phosphoglycerate kinasegenes may be used similarly to express the cloned LDH gene in Kmarxianus.

Example 6 Amplification of Linear Fragments of Homologous DNA for GeneDisruptions of Pyruvate Decarboxylase

[0150] An 82 bp oligomeric primer (5′kmPDC1Ko) was designed to contain51 bp identical to the 5′ end of the pyruvate decarboxylase (PDC) fromK. marxianus and 30 bp identical to the 5′ end of the ADH1 promoter frompHES vectors. The sequence of 5′KmPDC1Ko is as follows:5′-TAAACAGTACAATCGCAAAGAAAAGCTCCACACCCAAACCAAATAATTGCAATGCAACTTCTTTTCTTTTTTTTTCTTTTCT-3′ (SEQ ID NO: 7). The sequence forthe PDC genes from yeasts (K. marxianus or Y. stipitis or H. polymorpha)was obtained from the submitted Genbank sequence. Similarly, a reverse79 bp oligomer (3′kmPDC1Ko) was designed to contain 54 bp that wereidentical to the 3′ end of the PDC gene and 22 bp that were identical tothe 3′ end of the ADHI terminator. The sequence of 3′kmPDC1Ko is asfollows: 5′-TTATAAAATCATTAAAATCCAAAATCGTAATTTATCTCTTTATCCTCTCCCTCTCTACATGCCGGTAGAGGTGTGGTCA-3′ (SEQ ID NO:8). The primers weredesigned to amplify a linear DNA fragment from the pLh-ldh-HES andpPa-ldh-HES plasmids wherein the fragment contained the entire lactatedehydrogenase gene along with the ADH1 promoter and terminator (FIG. 6).The PCR amplified product also contains ends that were homologous tosequences from either K. marxianus PDC1, Yamadazyma stipitis PDC1 andPDC2, and Hansenula polymorpha PDCI and PDC2. The amplification reactionwas performed using Pfu DNA polymerase (New England Biolabs; Beverly,Mass.). 100 ng of pLh-ldh-HES or 5 pPa-ldh-HES was used in the reactionalong with 5 units of polymerase and 100 nmoles of the oligomers. Thereaction was carried out according to protocols described in Sambrook etal. (ibid.). FIG. 6 depicts the final linear product with the describedhomologies.

[0151] Alternate constructs were prepared to improve the likelihood ofobtaining a pdc negative strain of K. marxianus. To prepare theseconstructs, a 5.5 kbp fragment surrounding the K. marxianus 1.7 kbp PDC1gene was isolated (FIG. 6b) using PCR and genome walking techniques(Clonetech). The 5.5 kbp fragment was then cloned into the pCRII TAcloning vector (Invitrogen) using standard methods. A portion ofapproximately 370 bp near the middle of the 1.7 kbp coding region ofPDCI was removed from the K. marxianus 5.5 kbp fragment by restrictiondigestion (Sambrook et al., ibid.). The removed fragment has thefollowing sequence: CCGGTTCTTTCTCTTACTCTTACAAGACCAAGAACATTGTCGAATTCCACTCCGACTACATCAAGGTCAGAAACGCCACTTTCCCAGGTGTCCAAATGAAGTTCGTCTTGCAAAAGTTGTTGACCAAGGTCAAGGATGCTGCTAAGGGTTACAAGCCAGTTCCAGTTCCTCACGCTCCAAGAGACAACAAGCCAGTTGCTGACTCTACTCCATTGAAGCAAGAATGGGTCTGGACTCAAGTCGGTAAGTTCCTACAAGAAGGTGATGTTGTTCTAACTGAAACCGGTACCTCCGCTTTCGGTATCAACCAAACCCACTTCCCAAATGACACCTACGGTATCTCCCAAGTCTTGTGGGGTTCCATTGGTTTCA (Sequence ID No. 10).

[0152] A kanamycin resistance gene and its promoter was then isolatedfrom a pPIC9K vector (Invitrogen) using standard restriction technology(See Sambrook et al.), and cloned into the site in the 5.5 kbp fromwhich above-identified fragment was removed. The pPIC9K (Invitrogen)kanamycin resistance gene and its promoter were inserted such that thesequence of the inserted region was as follows: (SEQ ID NO:9)GTACAACTTGAGCAAGTTGTCGATCAGCTCCTCAAATTGGTCCTCTGTAACGGATGACTCAACTTGCACATTAACTTGAAGCTCAGTCGATTGAGTGAACTTGATCAGGTTGTGCAGCTGGTCAGCAGCATAGGGAAACACGGCTTTTCCTACCAAACTCAAGGAATTATCAAACTCTGCAACACTTGCGTATGCAGGTAGCAAGGGAAATGTCATACTTGAAGTCGGACAGTGAGTGTAGTCTTGAGAAATTCTGAAGCCGTATTTTTATTATCAGTGAGTCAGTCATCAGGAGATCCTCTACGCCGGACGCATCGTGGCCGACCTGCAGGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTTTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCTGCAGGTCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATG.

[0153] The resulting construct contains the G418 resistance genesurrounded by approximately 5 kbp of the pdc region as shown in FIG. 6c.A similar DNA construct was made which contained the internal G418 genesurrounded by 2.3 kbp of K. marxianus PDC1 in the pCRII vector as shownin FIG. 6d.

Example 7 Use of Linear DNA Fragment to Disrupt the Endogenous PDCCoding Sequence and Insert an LDH Coding Sequence Simultaneously

[0154] The linear DNA fragment generated by PCR described in Example 5is used to transform K. marxianus, Yamadazyma stipitis, or Hansenulapolymorpha. The protocol used for transformation is as described byWesolowski-Louvel et al. (NONCONVENTIONAL YEASTS IN BIOTECHNOLOGY:KLUYVEROMYCES LACTIS, ed. Klaus Wolf, Springer Verlag, Berlin, p.138-201 (1996)). Briefly, 5 mL of an overnight culture is spun down andwashed with electroporation buffer (10 nM Tris-HCl, 270 nM sucrose, 1 nMMgCl₂, pH 7.5). The washed cells then are incubated for 30 minutes at30° C. in incubation buffer (5 g/L yeast extract, 10 g/L peptone broth,10 g/L glucose, 25 nM DTT, 20 nM HEPES, pH 8.0). At the end of thisperiod, the cells are washed again and resuspended in 400 uL incubationbuffer. DNA (200 ng) is added to these cells, and the cells are pulsedusing the Bio-Rad Gene Pulser at 1800 volts, 1000 Cl, and 25 μF in a 0.4cm cuvette.

[0155] The cells are plated on regular YPD (10 g/L yeast extract, 20 g/Lpeptone broth, 20g/L glucose, 15% agar plates, and colonies are allowedto regenerate over 72 hours. Each of the plates with colonies arereplica plated on fresh YPD plates and incubated for 48 hours. Thecolonies then are overlayed with 6.5% soft agar (0.5% agar in 300 mMTris-HCl, 187 mM glutamate, pH 8.3). A staining mixture (3.2 mL of 1%agar, 1.6 mL of 120 mM Tris, 75 mM glutamate, pH 8.3, 0.4 mL of 2 mg/mLphenazine methosulfate, 7 units of glutamate pyruvate transaminase, and7 units of L(+)-pig muscle lactate dehydrogenase) is added to theoverlayed plates.

[0156] Yeast strains with high L(+) form blue halos within 10-120minutes. This method is similar to the method suggested by Subden et al.(Canadian J. Microbiol., 28:883-886 (1982)), and modified by Witte etal. (J. Basic Microbial. 29:707-7 16 (1989)). The colonies are selected,and the DNA isolated from the colonies is tested by PCR analysis andsequenced to detect the disrupted pyruvate decarboxylase gene.

[0157] In another embodiment, the clones described in Example 6 aboveand depicted in FIGS. 6c and 6 d are digested with two restrictionenzymes (see, Sambrook et al., ibid.) to yield approximately 3micrograms of fragment DNA containing the homologous PDC region thatincludes the mid-sequence inserted kanamycin resistance gene. Kmarxianus is transformed with the fragment using known techniques, suchas electroporation, to disrupt the pdc of K. marxianus.

[0158] Generally, electroporation is performed as follows: a) grow aculture of the microorganism in YPAD overnight (˜15 h) in a volume of 20mL; b) transfer 500 uL from the culture to a microfuge tube, spin@4L 4mm, discard supernatant; c)wash the pellet with 1 mL cold EB(EB=Electroporation Buffer: 10 mM Tris-HCl, pH 7.5; 270 mM Sucrose; 1 mMMgCl₂.); d) resuspend in 1 mL IB (IB=Incubation Buffer: YPD; 25 mM DTT;20 mM HEPES, pH8.0.); e) shake@800 rpm, 30° C. for 30 min in anEppendorf Thermomixer; f) spin down, wash once with EB, resuspend in 400uL EB; g) add three micrograms fragment DNA (in water 10 mM Tris-Cl, pH8.5), incubate on ice 30 mm; h) transfer to 0.4 cm electroporationcuvette. Bio-Rad Gene Pulser settings: 1000V, 1000 Cl, 50 TF. Timeconstant after pulse: ˜20 msec; i) transfer to 3 mL Morton Closure tube,incubate without shaking at 30° C. for 1 hour. Add 400 uL liquid YPADmedia (YPAD: 10 g Yeast Extract; 20 g Peptone; 20 g Glucose; 100 mgAdenine Hemisulphate. Volume=1 L. No pH adjustment), shake@800 rpm, 30°C. for 1 hour in Eppendorf Thermomixer. Add 400 uL liquid YPAD andrecover 4-6 hours; j) spin down in microfuge tube 4K, 4 mm, discardsupernatant, resuspend in 400 uL 1M Sorbitol; k) plate onto 200 ug/mlG418 selective plates; and 1) incubate at 30° C. for three to five days.

[0159] The colonies are screened first by a second patching onto 300ug/ml G418. The genomic DNA is isolated from the secondary yeast patchby standard genomic preparations (Sambrook). These are then screened viaPCR for 1) the presence of the kanamycin fragment using suitable primersand conditions (Sambrook) and 2) the absence of the disrupted pdc regionusing suitable primers and PCR conditions.

[0160] Colonies positive for the selection marker and negative for thepdc disruption region were then grown and analyzed by HPLC forphysiology. Genomic DNA from those strains was further analyzed bysouthern hybridization analysis.

Example 8 Growth Characteristics of Cells

[0161] 1. Low pH/high Temperature

[0162] Overnight cultures of K marxianus were inoculated into 50 mLyeast minimal medium according to Kiers et al. (Yeast, 14(5):459-469(1998)). Glucose (100 g/L) was used as the carbon source. The overnightcultures were maintained at 40° C., and inoculated into medium that wasalso maintained at 40° C. Addition of the inoculant changed the pH ofthe medium from 5.5 to 2.5. During the experiment, the pH remained 2.5.Glucose concentration was measured by YSI-membrane, and optical density(OD) was measured using a spectrophotometer.

[0163] Glucose was utilized in 72 hours, indicating that metabolicactivity occurs under low pH and high temperature culture conditionsduring that time period (FIG. 7). In addition, biomass decreasedslightly during the 48 to 72 time period, indicating that cellcatabolism out paces anabolism (FIG. 7).

[0164] 2. Pentose Carbon Sources

[0165] Overnight cultures of K. marxianus were inoculated into three 50mL flasks containing yeast minimal medium according to Kiers et al.(Yeast, 14(5):459-469 1998)). Each of the three flasks contained adifferent carbon source. The first contained 10 percent glucose, thesecond contained 10 percent D-xylose, and the third contained 10 percentL-arabinose. The flasks were incubated at 30° C., and the ODmeasurements were made periodically.

[0166] After 40 hours, the biomass yield for yeast cultured with glucoseor xylose was similar, while the biomass yield for yeast cultured witharabinose was lower (FIG. 8). Comparing the growth of yeast culturedwith glucose to those cultured with xylose or arabinose revealed aninitial lag time in growth. The yeast cultured with arabinose exhibiteda lag time of a few hours, while the lag time for yeast cultured withxylose was much more pronounced (FIG. 8). The presence of this lag tuneindicates that the yeast cells need time to adapt to the xylose andarabinose carbon sources. Presumably, this time is needed to induce thesynthesis of polypeptides not normally expressed.

[0167] 3. Corn Fiber Hydrolysate at Low pH

[0168] Overnight cultures of K. marxianus were inoculated into flaskscontaining yeast minimal medium according to Kiers et al. (Yeast,14(5):459-469 (1998)). Each flask contained 30% corn fiber hydrolysateas the carbon source. Briefly, the corn fiber hydrolysate was made byreacting corn fiber with 1.2% sulfuric acid at 145° C. for 25 minutes.During the reaction, the hemicellulose was broken down into themonomeric products arabinose, xylose, and glucose. Because of the hightemperature during the reaction, some arabinose and xylose was degradedinto furfural, while some glucose was degraded intohydroxymethlyfurfural. HPLC analysis of the hydrolysate revealed thepresence of 38.7 grams/L glucose, 39.1 grams/L xylose, 20.7 grams/Larabinose, and 1.6 grams/L furfural. In addition, the hydrolysate had apH of 1.51. Before culturing the yeast the pH of the corn fiberhydrolysate was adjusted to 3.0. During the culturing experiment, ODmeasurements were made periodically.

[0169] The yeast cells were capable of generating biomass when culturedwith corn fiber hydrolysate (FIG. 9).

[0170] 4. Various pH Conditions

[0171] Overnight cultures of K. marxianus were inoculated into fourflasks containing 50 mL yeast YPD medium (10 g/L yeast extract, 20 g/Lpeptone broth, 20 g/L glucose). Each flask had a different pH, which wasadjusted using HCl. During the culturing experiment, the temperature wasmaintained at 30° C., and OD measurements were made periodically. Growthwas observed within each flask (FIG. 10).

[0172] 5. Various pH Conditions/Lactic Acid

[0173] Overnight cultures of K. marxianus were inoculated into fourflasks containing 50 mL yeast YPD medium (10 g/L yeast extract, 20 g/Lpeptone broth, 20 g/L glucose) as well as 40 g/L lactic acid. Theaddition of the lactic acid resulted in a pH of 2.8. Thus, the pH withinthree flasks was adjusted to the indicated pH using NaOH. During theculturing experiment, the temperature was maintained at 30° C., and ODmeasurements were made periodically. Growth was observed within eachflask (FIG. 11).

Example 9 Recombinant Cells Capable of Producing acrylyl-CoA

[0174] An organism incapable of utilizing acrylate as a carbon source(e.g., E. coil) is transformed with a Clostridium propionicum genomicDNA library. The C. propionicum genomic library is generated using thepHES plasmid and expresses thereby 10 kbp fragments of the C.propionicum genome. The transformed E. coil are plated on selectionmedia with acrylic acid as the only carbon source. Only those cells thathave the ability to assimilate acrylate will grow. Acrylate is normallyassimilated by its enzyme-mediated conversion into lactate. In turn,lactate can be converted into pyruvate and utilized by the cells via theKrebs cycle.

[0175] Once a transformed E. coil is selected, the DNA plasmid from thegenomic library is isolated, and the inserted fragment sequenced. Oncesequenced, the fragment is scanned for open reading frames to determinethe coding sequence for enzymes involved in the conversion betweenlactate and acrylate (e.g., lactoyl-CoA dehydrogenase and CoAtransferases).

[0176] The isolated clones containing the coding sequence for theseenzymes is introduced into the yeast cells described in Example 6, whichcontain lactate dehydrogenase and lack pyruvate decarboxylase activity.Selection of recombinant yeast cells that contain the introduced nucleicacid is performed using G418 (300 g/L). Once isolated, the recombinantyeast cells are grown aerobically on glucose, and then switched toanaerobic conditions. The broth then is collected and assayed toacrylate using standard HPLC methods as described by Danner et al.(Biotechnological production of acrylic acid from biomass, In: AppliedBiochemistry and Biotechnology, Vol. 70-72 (1998)).

Example 10 Recombinant Cells Capable of Producing Ascorbate

[0177] Expression vectors are engineered such that the followingpolypeptides are expressed: 2,5-dioxovalerate dehydrogenase,5-dehydro-4-deoxy-D-glucarate dehydratase, glucarate dehydratase,aldehyde dehydratase, glucuronolactone reductase, and L-gluonolactoneoxidase. The nucleic acid sequence encoding these polypeptides areisolated from various microorganisms. Once engineered, the expressionvectors are transformed into a yeast cells by electroporation. Oncetransformed, the yeast cells are analyzed to determine whether or notthey produce L-ascorbate.

Example 11 Recombinant Cells Capable of Producing D-xylose

[0178] Expression vectors are engineered such that the followingpolypeptides are expressed: 2-dehydro-3-deoxy-D-pentanoate aldolase,xylonate dehydratase, xylonotactonase, and D-xylose dehydrogenase. Thenucleic acid sequences encoding these polypeptides are isolated fromPseudomonas spp. Once engineered, the expression vectors are transformedinto yeast cells by electroporation. Once transformed, the yeast cellsare analyzed to determine whether or not they produce D-xylose or otherpentose carbon compounds.

Example 12 Recombinant Cells Capable of Producing Citrate

[0179] PCR primers are designed based on the S. cerevisiae aconitase(ACOI, Genbank accession number M33 13 t) nucleic acid sequence. Theseprimers are used to clone the aconitase encoding nucleic acid from aKluyveromyces, Yamadazyma, or Hansenula species. Once sequenced, linearconstructs are made as described in Example 5, and used to disrupt theaconitase encoding nucleic acid within yeast cells. The selection markerused is the antibiotic G418 instead of lactate production as describedin Example 5. The nucleic acid providing resistance to antibiotic G418is the neomycin/kanamycin gene. This gene is obtained from the pPIC9Kvector (InVitrogen), and inserted into the pHES vector. Yeast cells aretransformed with PCR generated linear fragments that are engineered tohave ends homologous to the ACO1 as described above. The linear fragmentis designed to encode the G418 resistance gene. Only cells that haveintegrated the linear fragment in the location of the aconitase encodingnucleic acid are resistant to the antibiotic. Those cells are analyzedfor the appropriate integration using PCR. The yeast cells obtained bythis method have a partially functional TCA cycle, and thus canoverproduce citrate.

[0180] The citrate is transported across the mitochondrial membrane andinto the broth. In addition, these yeast cells are given an exogenousnucleic acid molecule that encodes an enzyme such as ATP-citrate lyasesuch that they can catalyze the conversion of accumulated citrate intooxaloacetate (see Example 13).

Example 13 Recombinant Cells Capable of Expressing Citrate Lyase in theCytosol

[0181] A crabtree positive yeast cell is transformed with the pHESplasmid containing a nucleic acid sequence that encodes a polypeptidehaving ATP-citrate lyase activity. This nucleic acid is isolated from E.coil, Krebsiella pneumoniae (Genbank accession number X798 17), or otherpublished sources. Once transformed, the yeast cells are analyzed todetermine whether or not they can utilize sugars to produce largeamounts of lipid accumulation. In addition, the yeast cells are analyzedto determine whether or not they exhibit ATP-citrate lyase activity asdescribed by Holdsworth et al. (J. Gen. Microbiol.,134:2907-2915(1998)). The yeast cells having ATP-citrate lyase activityare capable of providing cytosolic acetate under aerobic conditions by aroute other than the breakdown of aldehyde to acetate via aldehydedehydrogenase. In addition, when such yeast lack pyruvate decarboxylaseor aldehyde dehydrogenase activity, they should be able to provideacetate for biosynthesis via the Krebs cycle.

Example 14 Recombinant Cells Unable to Utilize Lactate as Carbon Source

[0182] Yeast cells are engineered such that the activity of a carboxylicacid transporter similar to the S. cerevisiae JENI polypeptide isreduced. Such yeast cells will have a reduced ability to transportlactate, and hence utilize lactate less efficiently. The activity of thecarboxylic acid transporter within yeast cells is reduced by disruptingthe locus containing the coding sequence for this polypeptide. First,the homologue of the JEN1 polypeptide is isolated from a host cell usingdegenerate primers designed based on the available sequence for JEN1(Genbank accession number U24 155). Once the nucleic acid is isolatedfrom the host cell, it is sequenced. Disruption of the coding sequencefor this polypeptide is done using the procedures described in Example11. Linear fragments are generated encoding homologous regions to theJEN1 sequence as well as the entire G418 resistance gene. This linearfragment is integrated into the JEN1 genomic sequence causing disruptionof the activity. Cells lacking carboxylic acid transporter activity areidentified by their inability to transport carboxylic acid, and hencetheir inability to grow when cultured on lactate.

[0183] In addition, cells are modified such that the activity of afunctional equivalent of the S. cerevisiae cytochrome b2 polypeptide isreduced. The cytochrome b2 polypeptide enables S. cerevisiae cells tometabolize lactate within the mitochondria. First, degenerate primersare -designed from the Saccharomyces cytochrome b2 sequence (Genbankaccession number Z46729). Once isolated, the clone is sequenced. Thedisruption of the yeast host homologue of cytochrome b2 is done usingmethods described in Methods in Yeast Genetics (Eds. Alison et al., ColdSpring Harbor Press (1997)). This recombinant yeast cell will be unableto utilize lactate as a carbon source.

Example 15 Large-Scale Production of Lactate

[0184] Multiple variants of K. marxianus cells having reduced PDCactivity are produced and isolated. Each variant is engineered tocontain a different copy number of an exogenous nucleic acid moleculeencoding a polypeptide having LDH activity. The LDH polypeptide is frommultiple different sources. Such variant cells can have differentspecific productivity for lactic acid at 40° C.

[0185] Each variant is grown in a vessel under aerobic conditions withan air flow of 1.5 VVM and a dissolved oxygen content of 30% to reach acell density of about 60 g/L, dry basis. Once the density is sufficient,the air flow is turned off, and the conditions within the vessel areswitched to anaerobic conditions. No base is added.

[0186] The variants with the highest specific productivity during theanaerobic phase can be found not only to produce lactic acid faster, butalso to achieve a higher concentration at a lower pH, than the variantswith lower specific productivity.

[0187] Product yield on glucose during the production phase can exceed90%. Certain variants are selected and subjected to the same culturingmethods except that the air flow is reduced to 0.1 VVM, rather thanbeing completely shut off Under such conditions, the final pH within thevessel can be lower, and the lactate concentration can be higher thanthe conditions with no air flow. Product yield on glucose can be reducedbut can remain at about 90%. When the test is repeated, hut with an airflow of 0.5 VVM, the product yield on glucose can be reduced to lessthan 80%.

Example 16 Large-Scale Production of Lactate Using a Series of BatchFermentations

[0188] A culture of K. marxianus lacking PDC activity and having LDHactivity is used as the inoculum in a series of batch fermentations.Each fermentation is carried out in progressively larger vessels, eachof which is sterilized immediately prior to use. In addition, eachvessel is provided with an air flow of 1.5 VYM and stirring sufficientto maintain a dissolved oxygen content above 10%. The final vessel has avolume of 6,000 L. The vessels also are maintained at a temperature of45° C. to enhance survival of the genetically modified K. marxianuscells over wild-type yeast and other microorganisms. Each vessel isfilled with standard culture medium for optimal growth.

[0189] The contents of the final vessel, with a cell density of 100grams of cells/L, dry basis, are transferred to a recently steamedproduction vessel having a volume of 300,000 L. Optionally, additionalcells obtained from the filtration of a previous production process areadded. The cell density in the production vessel is 6 grams of cells/L,dry basis. Glucose is added to a level of 80 g/L. The conditions withinthe vessel are anaerobic with the temperature being 42° C. for a periodof 25 hours. The specific productivity is greater than 0.5 gramslactate/(gram biomass * hour) until near the end of the process, atwhich time the productivity begins to drop. Once productivity begins todrop, the cells are removed and saved for reuse. The final lactateconcentration can be 75 g/L with the pH being 2.8. After biomassremoval, the solution is concentrated by evaporation to a concentrationof 50% lactate. The free acid (about 86% of total lactate) is extractedby liquid extraction into an organic and back extracted at a highertemperature into water. The raffinate containing the lactate salt iseither cleaned and recycled as a buffer in the growth vessel, oracidified with, for example, sulfuric acid and purified.

Example 17 Comparison of Aerobic Production of a Crabtree Negative (K.marxianus) and a Crabtree Positive (S. uvarum) Organisms

[0190] A crabtree negative (K. marxianus) and a crabtree positive (S.uvarum) organism were each grown in aerobic and anaerobic batchfermenters. Batch cultivation was performed at 30° C. in laboratoryfermenters with a working volume of 1.5 L. The pH was maintained at5.0±0.1 by automated addition of 2 mol/L potassium hydroxide (KOH). Thefermentor was flushed with air (aerobic cultures) or nitrogen gas(anaerobic cultures) at a flow rate of 0.8 L/min and stirred at 800 rpm.The dissolved-oxygen concentration was continuously monitored with anoxygen electrode (Ingold, type 34 100 3002). in the aerobic cultures,the dissolved oxygen concentration was maintained above 60%. Ten mLsamples were withdrawn at appropriate intervals for determination of dryweight and metabolite concentrations. Tween-80 and ergosterol were addedto anaerobic cultures to supply the compounds required for fatty acidsynthesis.

[0191] During exponential growth, both the dry weight and OD660 of yeastcultures, and the glucose and ethanol concentration in the supernatantwere determined at appropriate intervals. The specific ethanolproduction rate (q_(ethanol) mmol/g * h) was calculated by the followingequation using linear regression analysis:

q _(ethanol=() dE/dC _(x))*μ_(max)

[0192] where dE/dt (the rate of increase of the ethanol concentration inthe culture; mmol/l 8 h) and dC_(x)/dt (the rate of increase of thebiomass concentration; g/l * h) were calculated using differentiation ofplots of ethanol concentration and biomass concentration versus time,μ_(max) (h⁻¹). The maximum specific growth rate on glucose was estimatedfrom the exponential part of a plot of C_(x) versus time. To calculatethe specific glucose consumption rate (q_(glucose), mmol/g * h), dE wasreplaced by dG (the amount of glucose consumed per hour).

[0193] In the aerobic hatch cultures, the Kluyveromyces and theSaccharomyces strains exhibited a maximum specific growth rate onglucose of 0.4 h⁻¹ and 0.28 h⁻¹, respectively. The high glucoseconcentration and the resulting high specific growth rate of theSaccharomyces culture resulted in high rates of aerobic alcoholicfermentation (Table 3, FIG. 1). The specific rate of glucose consumptionwas about 2-fold higher in the Saccharomyces strain compared to theKluyveromyces strain due to the vigorous alcoholic fermentation. From anenergetic standpoint, alcoholic fermentation is a less efficient way forthe cell to generate ATP. The biomass yield on glucose was 0.38 g/g forKluyveromyces and 0.14 g/g for the Saccharomyces uvarum. The ethanolyield on glucose was zero for the crabtree-negative phenotypeKluyveromyces strain and 1.83 mmol/mmol for the Saccharomyces, thecrabtree-positive phenotype, culture. TABLE 3 Maximum specific growthrate, specific rates (q, mmol (g biomass)⁻¹ h⁻¹) of ethanol productionand glucose consumption, the biomass yield (g/g), product yield(mmol/mmol), and carbon recovery (in %; only calculated for anaerobiccultures) during exponential growth in batch cultures of Saccharomycesuvarum and Kluyveromyces marxianus on mineral medium containing 2%(wt/vol) glucose. K. marxianus S. uvarum aerobic anaerobic aerobicanaerobic T_(max) (h⁻¹) 0.38 0.09 0.28 0.12 q_(glucose) 5.8 7.6 10.9 7.2q_(ethanol) 0 9.9 20 9.7 Y_(p/s) 0 1.3 1.83 1.35 Y_(x/s) 0.38 0.07 0.140.09 C-rec — 84.6 — 73.3

[0194] In anaerobic batch cultures, the specific growth rate and biomassyield for both strains was very low compared to that found under aerobicconditions (Table 3, FIGS. I and 2). For the Kluyveromyces and theSaccharomyces strains, the biomass yield was 0.07 and 0.09 g/g,respectively. Both the strains perform equally well with respect to thespecific rate of alcoholic fermentation under anaerobic conditions. Thiswas confirmed using CO₂ production data.

[0195] Generally, this Example demonstrates that aerobic production ofbiomass is much faster than anaerobic, and that yield of biomass underaerobic conditions is higher for crabtree negative organisms (because,in crabtree positive organisms, some alcoholic fermentation takes place,using up glucose). This Example also demonstrates that the fermentationproduct (ethanol, in this case) is produced at the same rate for bothcrabtree positive and negative organisms under anaerobic conditions.Thus, an aerobic growth stage provides the high biomass yield, and asubsequent anaerobic fermentation stage channels metabolic energy intoproduct formation (rather than more growth). Overall, a process in whichproduction is separated from growth provides greater process flexibilityand better control over the overall process yield.

Example 18 Improved Lactate Production in a Host Strain that NaturallyMakes L-lactic Acid: Amplification of Linear Fragments of Homologous DNAfor Gene Disruptions of Pyruvate Decarboxylase

[0196] The yeast Kluyveromyces thermotolerans (K. thermotolerans) is anatural producer of L-lactic acid (Kurtzman and Fell, (1998) “TheYeasts, A Taxonomic Study” pp. 240-241; Elsevier Science B.V.;Amsterdam, The Netherlands). K. thermotolerans has a naturally occurringlactate dehydrogenase (ldh) gene that allows for the production ofL-lactic acid. The amount of lactic acid produced under anaerobicconditions is approximately 4% g/g of glucose utilized, while theremainder of the glucose is essentially converted into ethanol (42,5%g/g glucose consumed), glycerol (3% g/g of glucose consumed) and acetate(0.3 g/g % of glucose consumed). TABLE 4 Results of anaerobicfermentation using K. thermotolerans, starting with 100 g/l glucose inYPAD media (rich media). Time Glucose Lactic Acetate Glycerol EthanolLactic YSI 0 92.937 0 0 0 0.025 0.06 12 76.603 0.476 0 0.41 3.345 0.6 3638.618 2.135 0 2.011 25.642 2.08 54 11.662 3.525 0.2 2.789 41.522 3.3478 1.539 4.322 0.209 3.213 42.5 3.88 98 0.286 4.365 0.307 3.24 42.5 3.74

[0197] A 600 bp region of the PDC1 was isolated from K. thermotoleransusing consensus primers constructed from a sequence derived by comparingthe PDC1 gene sequence from K. marxianus and K. lactis. The PDCIfragment was then sequenced (Sanger), and used to isolate a 7.5 kbpfragment surrounding the K. thermotolerans pdcl (FIG. 6e) using PCR andgenome walking techniques (Clonetech). The 7.5 kbp fragment was thencloned into the pCRII TA cloning vector (Invitrogen). A portion ofapproximately 730 bp near the middle of the coding region of PDCI wasremoved from the K. thermotolerans 7.5 kbp fragment. The portion of K.thermotolerans pdcl removed by restriction digests (Sambrook) containedthe following sequence:

[0198] TTACCACTGTCTTCGGTCTGCCAGGTGACTTCAATCTGCGTCTGTTGGAC

[0199] GAGATCTACGAGGTCGAGGGTATGAGATGGGCCGGTAACTGTAACGAGT

[0200] TGAACGCTTCTTACGCTGCCGACGCTTACGCCAGAATCAAGGGTATGTCC

[0201] TGTTTGATCACCACCTTCGGTGTCGGTGAGTTGTCCGCTTTGAACGGTAT

[0202] CGCCGGTTCTTACGCTGAGCACGTCGGTGTCTTGCACATTGTCGGTGTCC

[0203] CATCCGTCTCCGCCCAGGCCAAGCAGCTATTGTTGCACCACACCTTGGGT

[0204] AACGGTGACTTCACTGTCTTCCACAGAATGTCCGCCAACATCTCTGAGAC

[0205] CACTGCTATGATCACTGATCTAGCTACCGCCCCATCTGAGATCGACAGAT

[0206] GTATCAGAACCACCTACATTAGACAGAGACCTGTCTACTTGGGTTTGCCA

[0207] TCTAACTTCGTTGACCAGATGGTCCCAGCCTCTCTATTGGACACCCCAAT

[0208] TGACTTGGCCTTGAAGCCAAACGACCAGCAGGCTGAGGAGGAGGTCATC

[0209] TCTACTTTGTTGGAGATGATCAAGGACGCTAAGAACCCAGTCATCTTGGC

[0210] TGACGCTTGCGCTTCCAGACACGATGTCAAGGCTGAGACCAAGAAGTTG

[0211] ATTGACATCACTCAGTTCCCATCTTTCGTTACCCCAATGGGTAAGGGTTC

[0212] CATTGACOAGAAGCACCCAAGATTCGGTGGTGTCTACGTCGGTACCTTGT

[0213] (Sequence ID No. 11). A gene encoding kanamycin resistance,including its promoter, was then isolated from pPTC9K vector(Invitrogen) by restriction digestion (Sambrook), and cloned into thesite in the 7.5 kbp that from which the 730 bp fragment was removed. Thesequence of the kanamycin resistance gene and its promoter from pPIC9K(Invitrogen) was as follows:

[0214] GTACAACTTGAGCAAGTTGTCGATCAGCTCCTCAAATTGGTCCTCTGTAA

[0215] CGGATGACTCAACTTGCACATTAACTTGAAGCTCAGTCGATTGAGTGAAC

[0216] TTGATCAGGTTGTGCAGCTGGTCAGCAGCATAGGGAAACACGGCTTTTCC

[0217] TACCAAACTCAAGGAATTATCAAACTCTGCAACACTTGCGTATGCAGGT

[0218] AGCAAGGGAAATGTCATACTTGAAGTCGGACAGTGAGTGTAGTCTTGAG

[0219] AAATTCTGAAGCCGTATTTTTATTATCAGTGAGTCAGTCATCAGGAGATC

[0220] CTCTACGCCGGACGCATCGTGGCCGACCTGCAGGGGGGGGGGGGGCGCT

[0221] GAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAAT

[0222] CGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCT

[0223] TTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGA

[0224] ACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAA

[0225] AAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGC

[0226] TCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCG

[0227] AGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATA

[0228] TTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAG

[0229] TTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCC

[0230] AACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAA

[0231] GTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAG

[0232] CTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGT

[0233] CATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCC

[0234] TGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAG

[0235] GAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATT

[0236] TTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGG

[0237] GGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATG

[0238] CTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCA

[0239] TCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAAC

[0240] AACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGA

[0241] TTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCA

[0242] TGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGG

[0243] CTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTT

[0244] CATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGA

[0245] CACAACGTGGCTTTCCCCCCCCCCCCTGCAGGTCGGCATCACCGGCGCCA

[0246] CAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGA

[0247] TCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGG

[0248] TGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATG

[0249] (Sequence ID No.12). The resulting construct includes thekanamycin resistance gene (G418) surrounded by approximately 6.8 kbp ofthe PDC region as shown in FIG. 6f. The construct depicted in FIG. 6f isdigested with two restriction enzymes (Sambrook) to yield approximately3 micrograms of fragment DNA containing the homologous PDC region andthe mid-sequence inserted kanamycin resistance gene. K. thermotoleransis transformed with the fragment using known transformation techniques,such as electroporation to disrupt the PDC of K. thermotolerans. Themethod of electroporation is as follows: a) grow culture in YPADovernight (˜15 h) in a volume of 20 mL; b) transfer 500 uL of culture toa microfuge tube, spin@4K, 4 mm, discard supernatant; c) wash with 1 mLcold EB.(EB Electroporation Buffer: 10 mM Tris-HCl, pH 7.5; 270 mMSucrose; 1 mM MgCl₂); d) resuspended in 1 mL IB (IB=Incubation Buffer:YPD; 25 mM DTT; 20 mM HEPES, pH8.0.); e) shake 800 rpm, 30° C. for 30 mmin an Eppendorf Thermomixer; f) spin down, wash once with EB, resuspendin 400 uL EB; g) add three micrograms fragment DNA (in water 10 mMTris-Cl, pH 8.5), incubate on ice 30 mm; h) transfer to 0.4 cmelectroporation cuvette. Bio-Rad Gene Pulser settings: 1000V, 1000A, 50TF. Time constant after pulse: ˜20 msec; i) transfer to 3 mL MortonClosure tube, incubate without shaking at 30° C. for 1 hour; j) add 400uL liquid YPAD media (YPAD: 10 g Yeast Extract; 20 g Peptone; 20 gGlucose; 100 mg Adenine Hemisulphate. Volume 1L. No pH adjustment),shake 800 rpm, 30° C. for 1 hour in Eppendorf Thermomixer; k) add 400 uLliquid YPAD and recover 4-6 hours; 1) spin down in microfuge tube@4K, 4mm. discard supernatant, resuspend in 400 uL IM Sorbitol and plate onto100 ug/ml G418 selective plates; and m) incubate at 30° C. for three tofive days.

[0250] Colonies are screened first by a second patching onto a culturedish containing 200 ug/ml 0418. The genomic DNA is isolated from thesecondary yeast patch using standard genomic preparations (Sambrook).The isolated genomic is then screened via PCR for 1) the presence of thekanamycin fragment using suitable primers and conditions (Sambrook); and2) the absence of the disrupted PDC region using suitable primers andPCR conditions. Colonies positive for the selection marker and negativefor the PDC disruption region are then grown for further study, forexample, genomic DNA from these strains is further analyzed by southernhybridization analysis.

Example 19 Cloning of Yeast, Fungal and Bacterial LDH Genes

[0251] LDH-encoding genes were isolated from two species of yeast,Kluyveromyces thermotolerans and Torulaspora pretoriensis. These specieswere known to produce lactic acid (Witte et al., 1989, J. BasicMicrobiol. 29: 707-716). All conventional procedures were performedaccording to procedures set forth in Sambrook et al., ibid., except asotherwise noted.

[0252]Kluyveromyces thermotolerans LDH

[0253]K. thermotolerans was obtained from the American Type CultureCollection (ATCC Accession #52709) and grown under standard conditions.Genomic DNA was purified from these cells using an Invitrogen “Easy-DNA”kit according to the manufacturer's protocol. Degenerate amplificationprimers were designed by reverse translating conserved regionsidentified in alignments of L-LDH encoding genes from Rhizopus oryzae,Homo sapiens, Drosophila melanogaster, Aribidopsis thaliana, andLactobacillus helveticus. Two degenerate oligonucleotides were usedsuccessfully in polymerase chain reaction (PCR) amplifications. Theseoligonucleotides were: EJP4 GTBATYGGYTCHGGTAC (SEQ ID No. 13) and EJP5SWRTCDCCRTGYTCACC. (SEQ ID No. 14)

[0254] PCR amplification reactions were performed using Perkin Elmerbuffer II (1.5 mM MgCl₂) and AmpliTaq Gold polymerase. Each reactioncontained K. thermotolerans genomic DNA at a concentration of 6 ng/uL,each of 4 dNTPs at a concentration of 0.2 mM, and each of the EJP4 andEJP5 primers at 1 uM. Reactions were performed according to thefollowing cycling conditions: an initial incubation for 10 min at 95°C., followed by 35 cycles consisting of 30 sec at 95° C., 40 sec. at 52°C., 40 sec at 72° C. A faint product fragment of 116 basepairs (bp) wasgel purified using conventional procedures, reamplified using the sameconditions and amplification conditions disclosed herein, cloned, andsequenced.

[0255] The resulting sequence could be translated into a polypeptidethat exhibited excellent homology to known L-LDH-encoding genes. Twonon-degenerate primers, EJP8 and EJP9, were designed based on thissequence: EJP8 GTACAGTTCTGGATACTGCTCG (SEQ ID No. 15) and ETP9ACAGGCATCGATGCTGTC. (SEQ ID No. 16)

[0256] A genomic DNA library was constructed using K. thermotolerans DNAby inserting random fragments generated by partial Sau3A digests of theDNA into the BamH1 site of plasmid Yep9T (ref). The complete K.thermotolerans LDH-encoding gene was isolated by PCR using primers EJP8and EJP9 in combination with primers in the vector arms. These vectorprimers were EJP10 CTACTTGGAGCCACTATCGAC (SEQ ID No. 17) and EJP11GTGATGTCGGCGATATAGG. (SEQ ID No. 18)

[0257] In these amplification reactions, the conditions were asdescribed above except that the extension time was increase to 2 min andthe annealing temperature was increased to 58° C. Amplification productswere cloned and sequenced and shown to contain the remainder of the K.thermotolerans LDH-encoding gene from based on homology to knownsequence.

[0258] Finally, the full-length gene was re-isolated directly from K.thermotolerans genomic DNA using a high-fidelity polymerase (Pfu) in PCRamplification reactions using primers EJP12 and EJP13: EJP12GATCTCCTGCTAAGCTCTTGC (SEQ ID No. 19) and EJP13 GCAGTTTTGGATATTCATGC.(SEQ ID No. 20)

[0259] These amplification reactions were performed as described above.The coding sequence of this independently generated PCR product agreedcompletely with the AmpliTaq Gold generated sequences. The nucleic acidsequence of the coding region of the K. thermotolerans LDH-encoding geneis presented below as SEQ ID No. 20.

[0260] The translation of the complete coding sequence showedsignificant sequence identity to L-LDH-encoding genes fromSchizosaccharomyces pombe (49.5%), Bacillus megaterium (45.1%),Lactobacillus helveticus (36.8%), cow (35.3%), and Rhizopus oryae(32.6%), among others. !Nucleotide Sequence of K thermotolerans lactatedehydrogenase: ATGTTCCAAG ATACAAAGTC TCAAGCAGTA AGAACTGATG CCAAAACAGTAAAAGTTGTG 60 (SEQ ID No. 21) GTAGTGGGAG TGGGAAGTGT TGGGTCTGCCACAGCGTATA CGTTGCTTCT CAGCGGCATC 120 GTTTCCGAGA TTGTCCTTAT CGACGTGAACAAAGACAAAG CAGAGGGTGA AAGCATGGAC 180 TTAAACCACG CAGCACCTTC AAATACAAGGTCTCGAGCGG GTGATTATCC TGACTGCGCT 240 GGCGCGGCCA TTGTTATTGT CACATGTGGGATTAACCAAA AAATGGACA AACAAGGATG 300 GATCTTGCTG CAAAAAATGC CAACATTATGCTGGAAATCA TCCCCAATGT TGCCAAATAT 360 GCTCCTGATA CCATCCTGCT TATTGCCACGAATCCTGTCG ATGTTTTGAC CTATATTAGC 420 TATAAGGCGT CAGGGTTTCC ACTAAGCAGAGTTATCGGCT CAGGTACAGT TCTGGATACT 480 GCTCGTTTTA AATACATCCT CGGAGAGCACTTCAAGATCT CATCGGACAG CATCGATGCC 540 TGTGTAATTG GAGAACATGG TGATTCGGGTGTGCCTGTCT GGTCTCTTAC CAACATCGAC 600 GGCATGAAGC TCCGGGATTA CTGCGAAAAAGCCAACCACA TATTTGATCA GAATGCGTTC 660 CATAGAATCT TTGAGCAAAC GCGAGACGCTGCTTACGATA TCATCAAGCG CAAAGGCTAT 720 ACTTCATATG GAATCGCAGC GGGATTACTTCGCATAGTAA AGGCGATTTT AGAGGATACA 780 GGATCCACAC TTACAGTTTC AACCGTTGGTGATTATTTTG GGGTTGAACA AATTGCTATA 840 AGCGTCCCTA CCAAACTCAA TAAAAGTGGGGCTCATCAAG TGGCTGAACT TTCACTCGAT 900 GAGAAGGAAA TAGAATTGAT GGAAAAATCAGCTAGTCAGA TCAAATCAGT GATTGAGCAT 960 CATGGAGATCAAT 972

[0261] !Amino Acid Sequence of K thermotolerans lactate dehydrogenase:Met Phe Gln Asp Thr Lys Ser Gln Ala Val Arg Thr Asp Ala Lys Thr (SEQ IDNo. 22) 1               5                   10                  15 ValLys Val Val Val Val Gly Val Gly Ser Val Gly Ser Ala Thr Ala            20                  25                  30 Tyr Thr Leu LeuLeu Ser Gly Ile Val Ser Glu Ile Val Leu Ile Asp        35                  40                  45 Val Asn Lys Asp LysAla Glu Gly Glu Ser Met Asp Leu Asn His Ala    50                  55                  60 Ala Pro Ser Asn Thr ArgSer Arg Ala Gly Asp Tyr Pro Asp Cys Ala65                  70                  75                  80 Gly AlaAla Ile Val Ile Val Thr Cys Gly Ile Asn Gln Lys Asn Gly                85                  90                  95 Gln Thr ArgMet Asp Leu Ala Ala Lys Asn Ala Asn Ile Met Leu Glu            100               105                 110 Ile Ile Pro AsnVal Ala Lys Tyr Ala Pro Asp Thr Ile Leu Leu Ile        115                 120                 125 Ala Thr Asn Pro ValAsp Val Leu Thr Tyr Ile Ser Tyr Lys Ala Ser    130                 135                 140 Gly Phe Pro Leu Ser ArgVal Ile Gly Ser Gly Thr Val Leu Asp Thr145                 150                 155                 160 Ala ArgPhe Lys Tyr Ile Leu Gly Glu His Phe Lys Ile Ser Ser Asp                165                 170                 175 Ser Ile AspAla Cys Val Ile Gly Glu His Gly Asp Ser Gly Val Pro            180                 185                 190 Val Trp Ser LeuThr Asn Ile Asp Gly Met Lys Leu Arg Asp Tyr Cys        195                 200                 205 Glu Lys Ala Asn HisIle Phe Asp Gln Asn Ala Phe His Arg Ile Phe    210                 215                 220 Glu Gln Thr Arg Asp AlaAla Tyr Asp Ile Ile Lys Arg Lys Gly Tyr225                 230                 235                 240 Thr SerTyr Gly Ile Ala Ala Gly Leu Leu Arg Ile Val Lys Ala Ile                245                 250                 255 Leu Glu AspThr Gly Ser Thr Leu Thr Val Ser Thr Val Gly Asp Tyr260                 265                 270 Phe Gly Val Glu Gln Ile AlaIle Ser Val Pro Thr Lys Leu Asn Lys        275                 280                 285 Ser Gly Ala His GlnVal Ala Glu Leu Ser Leu Asp Glu Lys Glu Ile    290                 295                 300 Glu Leu Met Glu Lys SerAla Ser Gln Ile Lys Ser Val Ile Glu His305                 310                 315                 320 Leu GluIle Asn

[0262]Torulaspora pretoriensis LDH

[0263] The L-LDH-encoding gene of T. pretoriensis was isolated in asimilar manner to the K. thermotolerans gene. The strategy was again toisolate a segment of the gene using PCR amplification of T. preoriensisgenomic DNA using degenerate primers and to isolate the remainder of thegene via PCR-based chromosome walking. T. pretoriensis was obtained fromthe American Type Culture Collection (ATCC Accession No. ______).Genomic DNA was purified from these cells using the Invitrogen“Easy-DNA” kit according to the manufacturer's protocol. Degenerateprimers were designed based on conserved sequences as described above.Two degenerate oligonucleotides were used successfully in polymerasechain reaction (PCR) amplifications: EJP1 GTYGGTGCHGGTGCHGTHGG (SEQ IDNo. 23) and EJP6 SWRTCDCCRTGYTCBCC (SEQ ID No. 24).

[0264] Thermocycling parameters and reaction conditions were asdescribed above, except that T. pretoriensis genomic DNA was used at aconcentration of 20 ng/uL. A strong product DNA fragment of 508 bp wascloned and sequenced. The resulting 169 amino acid translation productexhibited excellent homology to known L-LDH-encoding genes.

[0265] The remainder of the gene was isolated by “walking” in bothdirections from the known sequence. This was accomplished using aGenomeWalker kit (Clontech #K1807-1) and Advantage Genomic PolymeraseMix (Clontech #8418-1) according to the manufacturer's instructions.Four gene-specific nested primers were used in addition to the adaptorprimers provided in the kit. The gene-specific primers were: EJP20ATCCACAACAGCTTACACGTTATTGAG (SEQ ID No. 25) EJP21GTTTGGTTGCTGGAAGTGGTGTTGATAG (SEQ ID No. 26) EJP22AACATTGAATAGCTTGCTCAGGTTGTG (SEQ ID No. 27) and EJP23GATAATAAACGCGTTGACATTTCAGATG. (SEQ ID No. 28)

[0266] Products were cloned and sequenced and found to contain theremainder of the LDH-encoding gene from T. pretoriensis, based onhomology to known sequences. Translation of the complete coding sequenceshowed significant sequence identity to L-LDH-encoding genes fromSchizosaccharomyces pombe (48.8%), Bacillus megaterium (42%),Lactobacillus helveticus (38.2%), cow (34.9%), and Rhizopus oryzae(32.2%), among others. The nucleic acid sequence of the coding region ofthe T. pretoriensis LDH-encoding gene is presented below as SEQ ID No.28, and the predicted amino acid sequence of the LDH protein encodedtherein is identified as SEQ ID No. 29. !Nucleotide Sequence of T.pretoriensis lactate dehydrogenase: ATGCATAGAT GTGCTAAAGT GGCCATCGTCGGTGCCGGCC AAGTTGGATC CACAACAGCT 60 (SEQ ID No. 29) TACACGTTATTATTGAGTAG TTTGGTTGCT GAAGTGGTGT TGATAGATGT CGATAAAAGA 120 AAGGTCGAAGGCCAATTTAT GGATCTGAAC CACGCGGCTC CTTTAACGAA GGAGTCACGA 180 TTCAGTGCTGGGGACTATGA AAGTTGTGCT GATGCTGCGG TTGTAATCGT AACGGGCGGG 240 GCTAATCAGAAACCTGGTCA AACTAGAATG GAGCTAGCCG AGAGGAACGT TAAAATCATG 300 CAGGAAGTGATCCCTAAGAT TGTGAAATAC GCCCCCAACG CAATTTTGCT GATTGCAACA 360 AACCCTGTCGATGTACTTAC CTATGCTAGT TTGAAAGCGT CGGGATTCCC AGCAAGCCTT 420 GTTATTGGTTCTGGGACAGT TCTCGACTCT GCTCGTATAC AGCACAACCT GAGCAAGCTA 480 TTCAATGTTTCATCTGAAAG TGTCAACGCG TTTATTATCG GGGAACATGG TGACTCAAGT 540 GTGCCCGTCTGGTCGCTTGC TGAGATTGCC GGCATGAAAG TGGAGGATTA CTGTAGGCAG 600 TCCAAGAGAAAGTTTGACCC CAGCATTCTG ACCAAAATAT ATGAGGAGTC GCGTGACGCG 660 GCAGCCTACATCATAGAACG CAAAGGCTAT ACCAATTTCG GGATTGCAGC AGGTTTGGCT 720 AGGATAGTGAGAGCTATTCT GAGAGATGAA GGTGCCCTAT TAACTGTGTC TACTGTAGGT 780 GAGCACTTTGGCATGAAAGA TGTTTCATTG AGTGTTCCA CTAGGGTAGA CAGGAGCGGC 840 GCTCACCATGTCGTCGACCT TCTGCTAAAC GACAAGGAGC TGGAGCAAAT TAAAACATCT 900 GGAGCCAAGATAAAGTCAGC CTGTGATGAA CTTGGCATT 939

[0267] Amino Acid Sequence of T. pretoriensis lactate dehydrogenase:(SEQ ID No.30) Met His Arg Cys Ala Lys Val Ala Ile Val Gly Ala Gly GlnVal Gly 1               5                   10                  15 SerThr Thr Ala Tyr Thr Leu Leu Leu Ser Ser Leu Val Ala Glu Val            20                  25                  30 Val Leu Ile AspVal Asp Lys Arg Lys Val Glu Gly Gln Phe Met Asp        35                  40                  45 Leu Asn His Ala AlaPro Leu Thr Lys Glu Ser Arg Phe Ser Ala Gly    50                  55                  60 Asp Tyr Glu Ser Cys AlaAsp Ala Ala Val Val Ile Val Thr Gly Gly65                  70                  75                  80 Ala AsnGln Lys Pro Gly Gln Thr Arg Met Glu Leu Ala Glu Arg Asn                85                  90                  95 Val Lys IleMet Gln Glu Val Ile Pro Lys Ile Val Lys Tyr Ala Pro            100                 105                 110 Asn Ala Ile LeuLeu Ile Ala Thr Asn Pro Val Asp Val Leu Thr Tyr        115                 120                 125 Ala Ser Leu Lys AlaSer Gly Phe Pro Ala Ser Arg Val Ile Gly Ser    130                 135                 140 Gly Thr Val Leu Asp SerAla Arg Ile Gln His Asn Leu Ser Lys Leu145                 150                 155                 160 Phe AsnVal Ser Ser Glu Ser Val Asn Ala Phe Ile Ile Gly Glu His                165                 170                 175 Gly Asp SerSer Val Pro Val Trp Ser Leu Ala Glu Ile Ala Gly Met            180                 185                 190 Lys Val Glu AspTyr Cys Arg Gln Ser Lys Arg Lys Phe Asp Pro Ser        195                 200                 205 Ile Leu Thr Lys IleTyr Glu Glu Ser Arg Asp Ala Ala Ala Tyr Ile    210                 215                 220 Ile Glu Arg Lys Gly TyrThr Asn Phe Gly Ile Ala Ala Gly Leu Ala225                 230                 235                 240 Arg IleVal Arg Ala Ile Leu Arg Asp Glu Gly Ala Leu Leu Thr Val                245                 250                 255 Ser Thr ValGly Glu His Phe Gly Met Lys Asp Val Ser Leu Ser Val            260                 265                 270 Pro Thr Arg ValAsp Arg Ser Gly Ala His His Val Val Asp Leu Leu        275                 280                 285 Leu Asn Asp Lys GluLeu Glu Gln Ile Lys Thr Ser Gly Ala Lys Ile    290                 295                 300 Lys Ser Ala Cys Asp GluLeu Gly Ile 305                 310

[0268]B. megaterium LDH

[0269]B. megaterium DNA encoding the LDH gene was isolated as follows.B. megaterium was obtained from the American Type Culture Collection(ATCC Accession #6458) and grown under standard conditions. Genomic DNAwas purified from these cells using an Invitrogen “Easy-DNA” kitaccording to the manufacturer's protocol. Primers were designed on thebasis of the available sequence in Genbank for the L-LDH from B.megaterium (Genbank accession # M22305). PCR amplification reactionswere performed using Perkin Elmer buffer II (1.5 mM MgCl₂) and AmpliTaqGold polymerase. Each reaction contained B. megaterium genornic DNA at aconcentration of 6 ng/uL, each of 4 dNTPs at a concentration of 0.2 mM,and each of two amplification primers BM1270 and BM179 at aconcentration of 1 uM, where these primers have the sequence: BM1270CCTGAGTCCACGTCATTATTC and (SEQ ID No.31) BM179 TGAAGCTATTTATTCTTGTTAC.(SEQ ID No.32)

[0270] Reactions were performed according to the following themocyclingconditions: an initial incubation for 10 min at 95° C., followed by 35cycles consisting of 30 sec at 95° C., 30 sec. at 50° C., 60 sec at 72°C. A strong product fragment of 1100 base pairs (bp) was gel purifiedusing conventional procedures, cloned, and sequenced. The resultingsequence could be translated into a polypeptide that exhibited excellenthomology to known L-LDH-encoding genes.

[0271] The coding sequence for the B. megaterium LDH-encoding gene (SEQID No. 32) disclosed herein was operatively linked to a promoter fromthe phosphoglycerate kinase gene and a transcriptional terminator fromthe GAL10 gene, both from the yeast Saccharomyces cerevisiae. In makingthis construct, the following oligonucleotides were prepared and used toamplify the coding sequence from a plasmid containing an insert havingthe sequence identified as SEQ ID No. 29. Two oligonucleotide primers,Bmeg5′ and Bmeg3′, were designed based on this sequence to introducerestriction sites at the ends of the coding sequence of the gene: Bmeg5′GCTCTAGATGAAAACACAATTTACACC and (SEQ ID No.33) Bmeg3′ATGGATCCTTACACAAAAGCTCTGTCGC. (SEQ D No.34)

[0272] This amplification reaction was performed using dNTP and primerconcentrations described above using Pfu Turbo polymerase (Stratagene)in a buffer supplied by the manufacturer. Thermocycling was done byinitially incubating the reaction mixture for 3 min at 95° C., then by20 cycles of 30 sec at 95° C., 30 sec at 50° C., 60 sec at 72° C.,followed by a final incubation for 9 min at 72° C. The product wasdigested with restriction enzymes XbaI and BamHI and then ligated intothe XbaI and BamHI sites of plasmid pNC101 (NREL). This ligationresulted in the PGK promoter and GAL10 terminator becoming operablylinked (i.e., trascriptionally-active in a yeast cell) to the B.megaterium LDH coding sequence. Once the B. megaterium LDH had beenoperably linked to these transcription control sequence, the NotI-NotIfragment was excised and re-cloned into a vector capable of replicatingin Kluyveromyces species (plasmid pNC003, NREL). The resulting plasmidcontained the LDH-containing NotI-NotI fragment as well as a 4,756 bpsequence between the SphI sites from the K. lactis plasmid pKD1.

[0273] These plasmids are shown in FIG. 17.

[0274]Rhizopus oryzae LDH

[0275] L-LDH was isolated from Rhizopus oryzae as follows. Rhizopusoryzae cells were obtained from the American Type Culture Collection(ATCC Accession #9363) and grown under standard conditions. Genomic DNAwas purified from these cells using an Invitrogen “Easy-DNA” kitaccording to the manufacturer's protocol. Primers were designed on thebasis of the available sequence in Genbank for the L-LDH from R. oryzae(Genbank accession # AF226154). PCR amplification reactions wereperformed using Perkin Elmer buffer II (1.5 mM MgCl₂) and AmpliTaq Goldpolymerase. Each reaction contained R. oryzae genomic DNA at aconcentration of 6 ng/uL, each of 4 dNTPs at a concentration of 0.2 mM,and each of the amplification primers Ral-5′ and Ral-3′ primers at 1 uM.The amplification primers had the sequence: Ral-5′CTTTATTTTTCTTTACAATATAATTC and (SEQ ID No.35) Ral-3′ACTAGCAGTGCAAAACATG. (SEQ ID No.36)

[0276] Reactions were performed according to the following cyclingconditions: an initial incubation for 10 min at 95° C., followed by 35cycles consisting of 30 sec at 95° C., 30 sec. at 41° C., 60 sec at 72°C. A strong product fragment of 1100 base pairs (bp) was gel purifiedusing conventional procedures, cloned in TA vector (Invitrogen,Carlsbad, Calif.) and sequenced. The resulting sequence could betranslated into a polypeptide that exhibited excellent homology to knownRhizopus oryzae L-LDH-encoding gene sequence in Genbank (Accession #AF226154).

[0277] The coding sequence for the R. oryzae LDH-encoding gene disclosedherein was operatively linked to a promoter from the phosphoglyceratekinase gene and a transcriptional terminator from the GAL10 gene, bothfrom the yeast S. cervisiae. In making this construct, the followingoligonucleotides were prepared and used to amplify the coding sequencefrom the plasmid containing the Rhizopus LDH insert. Two oligonucleotideprimers, Rapgk5 and Papgk3′, were designed based on this sequence tointroduce restriction sites at the ends of the coding sequence of thegene. Rapgk5 GCTCTAGATGGTATTACACTCAAAGGTCG and (SEQ ID No.37) Papgk3GCTCTAGATCAACAGCTACTTTTAGAAAAG. (SEQ ID No.38)

[0278] This amplification reaction was performed using dNTP and primerconcentrations as described above using Pfu Turbo polymerase(Stratagene) in a buffer supplied by the manufacturer. Thermocycling wasdone by initially incubating the reaction mixture for 3 min at 95° C.,then by 20 cycles of 30 sec at 95° C., 30 sec at 53° C., 60 sec at 72°C., followed by a final incubation for 9 min at 72° C. The product wasdigested with restriction enzymes XbaI and then ligated into the XbaIsite of plasmid pNC101 (NREL).

[0279] This ligation resulted in the PGK promoter and GAL10 terminatorbecoming operably linked (i.e., trascriptionally-active in a yeast cell)to the R. oryzae L-LDH coding sequence. Once the R. oryzae LDH had beenoperably linked to these transcription control sequence, the NotI-NotIfragment was excised and re-cloned into a vector capable of replicatingin Kluyveromyces species (plasmid pNC003, NREL). The resulting plasmidcontained the LDH-containing NotI-NotI fragment as well as a 4,756 bpsequence between the SphI sites from the K. lactis plasmid pKD1.

[0280] G418 Resistance Marker Vectors Encoding an LDH Gene Isolated fromK. thermotolerns, R. oryzae or B. megaterium

[0281] The G418 antibiotic selection marker obtained from Invitrogen(Carlsbad, Calif.) was modified and operatively linked to a promoterfrom the pyruvate decarboxylase gene and a transcriptional terminatorfrom the GAL10 gene, both from the yeast S. cerevisiae. In making thisconstruct, the following oligonucleotides were prepared and used toamplify the coding sequence from the plasmid containing the G418resistance gene insert. Two oligonucleotide primers, G5′ and G3′, weredesigned based on this sequence to introduce restriction sites at theends of the coding sequence of the gene. G5′AAATCTAGATGAGCCATATTCAACGGGA and (SEQ ID No.39) G3′CCGGATCCTTAGAAAAACTCATCGAGCAT. (SEQ ID No.40)

[0282] These oligonucleotides were used to amplify the G418 gene frompPIC9K vector (Invitrogen, Carlsbad, Calif.). This amplificationreaction was performed using dNTP and primer concentrations describedabove using Pfu Turbo polymerase (Stratagene) in a buffer supplied bythe manufacturer. Thermocycling was done by initially incubating thereaction mixture for 3 min at 95° C., then by 20 cycles of 30 sec at 95°C., 30 sec at 50° C., 60 sec at 72° C., followed by a final incubationfor 9 min at 72° C. The product was digested with restriction enzymesXbaI and BamHI and then ligated into the XbaI and BamHI site of plasmidpNC104 (NREL)

[0283] The LDH gene from the B. megaterium, operatively linked to apromoter from the phosphoglycerate kinase gene and a transcriptionalterminator from the GAL10 gene, both from the yeast S. cerevisiae, wasintroduced into this vector at the SphI site at the 3′ end of the Gal10Terminator of the G418 gene. This essentially coupled the G418 selectionmarker gene and the B. megaterium LDH gene to give plasmid pCA5. PlasmidpCA5 was restriction digested to excise the 4 kilobasepair (Kbp)fragment that consisted of the G418 selection marker gene and the B.megaterium LDH gene. This 4 Kbp fragment was used to transform K.marxianus using chemical and electroporation methods discussed herein.

Example 20 Transforming Yeast with Novel LDHs

[0284] The coding sequence for the K. thermotolerans LDH-encoding gene(SEQ ID No. 20) disclosed herein was operatively linked to a promoterfrom the phosphoglycerate kinase gene and a transcriptional terminatorfrom the GAL10 gene, both from the yeast Saccharomyces cervisiae. Inmaking this construct, the following oligonucleotides were prepared andused amplify the coding sequence from a plasmid containing the insert:(SEQ ID No.41) EJP14 GCTCTAGAATTATGTTCCAAGATACAAAGTCTCAAG and (SEQ ID.No.42) EJP15 CCGGAATTCATCCTCAATTGATCTCCAGATGCTC,

[0285] This amplification reaction was performed using dNTP and primerconcentrations described above using Pfu Turbo polymerase (Stratagene)in a buffer supplied by the manufacturer. Thermocycling was done byinitially incubating the reaction mixture for 3 min at 95° C., then by20 cycles of 30 sec at 95° C., 40 sec at 60° C., 60 sec at 72° C.,followed by a final incubation for 9 min at 72° C. The product wasdigested with restriction enzymes Xbal and EcoRI and then ligated intothe XbaI and EcoRI sites of plasmid pNC101 (SOURCE). This ligationresulted in the PGK promoter and GAL10 terminator becoming operablylinked (i.e., trascriptionally-active in a yeast cell) to the K.thermotolerans LDH coding sequence. A NotI-NotI fragment of thisplasmid, containing the resulting fusion of promoter, K. thermotoleransLDH coding sequence and terminator, is disclosed herein identified asSEQ ID No. 43: GCGGCCGCGG ATCGCTCTTC CGCTATCGAT TAATTTTTTT TTCTTTCCTCTTTTTATTAA 60 CCTTAATTTT TATTTTAGAT TCCTGACCTT CAACTCAAGA GGGACAGATATTATAACATC 120 TGCACAATAG GCATTTGCAA GAATTACTCG TGAGTAAGGA AAGAGTGAGGAACTATCGCA 180 TACCTGCATT TAAAGATGCC GATTTGGGCG CGAATCCTTT ATTTTGGCTTCACCCTCATA 240 CTATTATCAG GGCCAGAAAA AGGAAGTGTT TCCCTCCTTC TTGAATTGATGTTACCCTCA 300 TAAAGCACGT GGCCTCTTAT CGAGAAAGAA ATTACCGTCG CTCGTGATTTGTTTGCAAAA 360 AGAACAAAAC TGAAAAAACC CAGACACGCT CGACTTCCTG TCTTCCTATTGATTGCAGCT 420 TCCAATTTCG TCACACAACA AGGTCCTAGC GACGGCTCAC AGGTTTTGTAACAAGCAATC 480 GAAGGTTCTG GAATGGCGGG AAAGGGTTTA GTACCACATG CTATGATGCCCACTGTGATC 540 TCCAGAGCAA AGTTCGTTCG ATCGTACTGT TACTCTCTCT CTTTCAAACAGAATTGTCCG 600 AATCGTGTGA CAACAACAGC CTGTTCTCAC ACACTCTTTT CTTCTAACCAAGGGGGTGGT 660 TTAGTTTAGT AGAACCTCGT GAAACTTACA TTTACATATA TATAAACTTGCATAAATTGG 720 TCAATGCAAG AAATACATAT TTGGTCTTTT CTAATTCGTA GTTTTTCAAGTTCTTAGATG 780 CTTTCTTTTT CTCTTTTTTA CAGATCATCA AGGAAGTAAT TATCTACTTTTTACAACAAA 840 TCTAGAATTA TGTTCCAAGA TACAAAGTCT CAAGCAGTAA GAACTGATGCCAAAACAGTA 900 AAAGTTGTGG TAGTGGGAGT GGGAAGTGTT GGGTCTGCCA CAGCGTATACGTTGCTTCTC 960 AGCGGCATCG TTTCCGAGAT TGTCCTTATC GACGTGAACA AAGACAAAGCAGAGGGTGAA 1020 AGCATGGACT TAAACCACGC AGCACCTTCA AATACAAGGT CTCGAGCGGGTGATTATCCT 1080 GACTGCGCTG GCGCGGCCAT TGTTATTGTC ACATGTGGGA TTAACCAAAAAAATGGACAA 1140 ACAAGGATGG ATCTTGCTGC AAAAAATGCC AACATTATGC TGGAAATCATCCCCAATGTT 1200 GCCAAATATG CTCCTGATAC CATCCTGCTT ATTGCCACGA ATCCTGTCGATGTTTTGACC 1260 TATATTAGCT ATAAGGCGTC AGGGTTTCCA CTAAGCAGAG TTATCGGCTCAGGTACAGTT 1320 CTGGATACTG CTCGTTTTAA ATACATCCTC GGAGAGCACT TCAAGATCTCATCGGACAGC 1380 ATCGATGCCT GTGTAATTGG AGAACATGGT GATTCGGGTG TGCCTGTCTGGTCTCTTACC 1440 AACATCGACG GCATGAAGCT CCGGGATTAC TGCGAAAAAG CCAACCACATATTTGATCAG 1500 AATGCGTTCC ATAGAATCTT TGAGCAAACG CGAGACGCTG CTTACGATATCATCAAGCGC 1560 AAAGGCTATA CTTCATATGG AATCGCAGCG GGATTACTTC GCATAGTAAAGGCGATTTTA 1620 GAGGATACAG GATCCACACT TACAGTTTCA ACCGTTGGTG ATTATTTTGGGGTTGAACAA 1680 ATTGCTATAA GCGTCCCTAC CAAACTCAAT AAAAGTGGGG CTCATCAAGTGGCTGAACTT 1740 TCACTCGATG AGAAGGAAAT AGAATTGATG GAAAAATCAG CTAGTCAGATCAAATCAGTG 1800 ATTGAGCATC TGGAGATCAA TTGAGGATGA ATTCGGATCC GGTAGATACATTGATGCTAT 1860 CAATCCAGAG AACTGGAAAG ATTGTGTAGC CTTGAAAAAC GGTGAAACTTACGGGTCCAA 1920 GATTGTCTAC AGATTTTCCT GATTTGCCAG CTTACTATCC TTCTTGAAAATATGCACTCT 1980 ATATCTTTTA GTTCTTAATT GCAACACATA GATTTGCTGT ATAACGAATTTTATGCTATT 2040 TTTTAAATTT GGAGTTCAGT GATAAAAGTG TCACAGCGAA TTTCCTCACATGTAGGGACC 2100 GAATTGTTTA CAAGTTCTCT GTACCACCAT GGAGACATCA AAAATTGAAAATCTATGGAA 2160 AGATATGGAC GGTAGCAACA AGAATATAGC ACGAGCCGCG GATTTATTTCGTTACGCATG 2220 CGCGGCCGC 2229

[0286] Once the K. thermotolerans LDH had been operably linked to thesetranscription control sequence, the NotI-NotI fragment was excised andre-cloned into a vector capable of replicating Kluyveromyces species(plasmid pNC003, Invitrogen, Carlsbad, Calif.). The resulting plasmidcontained the LDH-containing NotI-NotI fragment (SEQ ID No. 43) as wellas a 4,756 bp sequence between the SphI sites from the K. lactis plasmidpKD1 (Chen et al., 1986, Nucleic Acids Res. 14: 4471-4481). In addition,pNC003 carries the zeocin resistance gene under the control of the yeastTEF promoter (Hwang et al., 1993, EMBO J. 12: 2337-2348). Bothorientations of the NotI-NotI fragment were obtained and were termedpNC102 andpNC103.

[0287] These plasmids were introduced into K. marxianus and K. lactissubstantially as described below. Plasmids pNC102 and pNC103 wereintroduced into the yeast cells by the chemical transformation method.Plasmid pNC003, which does not contain an LDH-encoding gene, wasintroduced into yeast cells as a control. Transformants were selected onYPD plates containing 200 ug/mL zeocin and grew up after 2 days at 30°C. For the K. marxianus transformed cultures, only one transformant ofpNC003 and one of pNC 103 were obtained. In K. lactis transformedcultures, multiple transformants of each plasmid were obtained.

[0288] Transformants were then analyzed for their ability to produceL-lactic acid. Cultures (2 mL) of YPD broth containing 20 g/L glucoseand 300 ug/mL zeocin were inoculated directly from colonies on thetransformation plates. The cultures were incubated without shaking for52 hours at 30° C. At the end of that period, the cells were removed bycentrifugation and the culture supernatant was assayed for glucose andL-lactic acid using a YSI. The results of these assays are shown inTable 5 below. Both K. marxianus and K. lactis cells containing the K.thermotolerans LDH plasmid were able to produce L-lactic acid tosignificant levels, whereas the control cells containing the emptyvector produced no detectable L-lactic acid. Thus, K. thermotolerans LDHis clearly able to functions in other species of yeast to channel carboninto the production of lactic acid. TABLE 5 Glucose L-lactic acid HostPlasmid Transformant# g/l g/l K. lactis pNC003 1 10.0 0.01 K. lactispNC003 2 9.9 0.02 K. lactis pNC102 1 10.1 2.4 K. lactis pNC102 2 10.52.4 K. lactis pNC102 3 10.0 2.5 K. lactis pNC102 4 10.1 2.4 K. lactispNC102 5 10.6 2.4 K. lactis pNC103 1 11.0 2.2 K. lactis pNC103 2 10.02.4 K. lactis pNC103 3 9.3 2.5 K. lactis pNC103 4 11.0 2.4 K. lactispNC103 5 9.9 2.5 K. marxianus pNC003 1 0.01 0.02 K. marxianus pNC103 10.00 1.3 YPD only 19.0 0.02

Example 21 Lactic Acid Production from D-xylose in Kluyveromycesmarxianus

[0289] In order to demonstrate that sugars other than glucose could beused to produce lactic acid, xylose fermentation to lactic acid wasconducted in 250-mL baffled shake flasks by genetically engineeredstrains of Kluyveromyces marxianus derived from K. marxianus 1 (ATCCAccession No. 52486). More specifically, the three strains used in thisexample are as follows: (i) NC39: K. marxianus 1 carrying the multi-copyplasmid pNC7 that contains the Zeocin selection marker on plasmid pNC3and the Bacillus megaterium lactate dehydrogenate (LDH) under control ofa phosphoglycerate kinase promoter as described below; (ii) NC103: K.marxianus 1 carrying the multi-copy plasmid pNC103 containing Zeocinselection marker on plasmid pNC003 and the Kluyveromyces thermotoleransLDH under control of a phosphoglycerate promoter as described above; and(iii) NC102: K. marxianus 1 carrying the multi-copy plasmid pNC102containing the Zeocin marker on a pKD1 vector. The latter strain wasused as the control. Presence of Zeocin in the media minimized plasmidloss.

[0290] The innoculum was prepared by transferring a single colony into a10 mL tube that contained 3 mL of defined complete medium supplementedwith 300 μg/mL Zeocin. The medium used in all experiments contained (perliter): 6.7 g Yeast-Nitrogen-Base (YNB; without amino acids and ammoniumsulfate), 3 g urea and 0.3 g Zeocin. As a carbon and energy sourceeither 20 g/L D-glucose or D-Xylose were added. The initial pH wasadjusted to 5.0 with potassium hydroxide. Cells were grown overnight ona rotary shaker at 250 rpm and 30° C. and then transferred to a 250 mLbaffled shake-flask containing 100 mL of the above described YNB media.Cells were again grown overnight at pH 5.0 and 30° C. and subsequentlystored in suspension in 15% (w/v) glycerol at −80° C. These stockcultures were used as an inoculum for the experiments described below.

[0291] Lactic Acid Production on Glucose with NC39 and NC 103

[0292] Cultures of strain NC102 NC 103 and NC39 were grown to stationaryphase on 2% (w/v) glucose in the above-described YNB media at 250 rpmand 30° C. After this, cells were pelleted and transferred to YNB mediasupplemented with 2% (w/v) glucose at an initial OD₆₀₀ of 20. The cellswere incubated at 100 rpm and 30° C. to reduce oxygen supply to theculture. Liquid samples were withdrawn from the culture at timeintervals to measure growth (using OD₆₀₀), metabolites and pH.Metabolite analysis was performed by HPLC using an Aminex HPX-87H column(operating at 55° C. with 10 mM H₂SO₄ as the mobile phase at a flow rateof 0.5 mL/min) hooked up to a Waters 410 Refractive Index detector. Whenthe pH dropped below 3.5, 2 g of sterile CaCO₃ was added to increase thepH to about 5.5.

[0293] Twenty-four hours after the transfer of cells to fresh medium,the glucose was fully depleted in all strains studied. In strains NC39and NC103, 6.4 g/L (32% yield) and 3.8 g/L (19% yield) lactic acid hadbeen produced, respectively. No lactic acid production could be detectedin the control strain NC102. Strain NC 39 and NC103 also produced 2.3and 2.9 g/L of ethanol, respectively; the control strain NC102 produced5.8 g/L of ethanol. No other typical fermentation products (pyruvate,succinic acid, glycerol and acetate) could be detected>1.0 g/L in allcultures.

[0294] These results established that Kluyveromyces marxianus (acrabtree-negative yeast strain) expressing a heterologous LDH (eitherfrom B. megaterium or K. thermotolerans) could be used to produce lacticacid from glucose. Lactic acid production on xylose from NC39 and NC103.

[0295] Lactic acid production using xylose as a carbon source wasdemonstrated using culture and fermentation conditions substantially asset forth above, except that glucose in the culture media was replacedby D-xylose. A maximum of 4.8 g lactic acid was produced from 20 gxylose (i.e., a yield of 0.23 g/g) in strain NC39 after 72 hoursfermentation. No lactic acid was produced in strain NC103 or controlstrain NC 102. In addition, no other fermentation products (such aspyruvate, acetate, glycerol, ethanol) could be detected at >1.0 g/L.

[0296] These results established that Kluyveromyces marxianus (acrabtree-negative yeast strain) expressing a heterologous LDH (eitherfrom B. megaterium or K. thermotolerans) could be used to produce lacticacid from xylose, albeit at a slower rate than with glucose.

[0297] These results are also shown in FIG. 15.

Example 22 L-Lactic Acid Production from Pentose Sugars in Yeasts

[0298] Candida species were genetically engineered to use pentose sugarsto produce lactic acid. Vectors and other constructs are shown in FIG.16.

[0299] Zeocin Resistance Vectors for C. sonorensis

[0300] The plasmid pTEF1/Zeo (Invitrogen) containing the zeocinresistance marker under control of S. cerevisiae TEF1 promoter wasmodified by adding a C. sonorensis rDNA fragment to provide a target forhomologous recombination. The following oligonucleotide primers: (SEQ IDNo.44) TGG ACT AGT AAA CCA ACA GGG ATT GCG TTA GT and (SEQ ID No.45) GTAGTC TAG AGA TCA TTA CGC CAG CAT CCT AGG,

[0301] which correspond to C. sonorensis 26 S rRNA (Genbank AccessionNo. U70185), were used to amplify C. sonorensis genomic DNA to provide aPCR-amplified fragment of the 26S rDNA gene. The resulting PCR productfragment was digested with restriction enzymes SpeI and XbaI and ligatedwith pTEF/Zseo plasmid digested with XbaI. The resulting plasmid,pMI203, is shown in FIG. 16.

[0302] The TEF1 promoter contained in pMI203 was replaced by a promoterof a gene from another Candida species, the C. albicans PGK1 promoter.The following oligonucleotide primers: (SEQ ID No.46) GCG ATC TCG AGGTCC TAG AAT ATG TAT ACT AAT TTG C and (SEQ ID No.47) ACT TGG CCA TGG TGATAG TTA TTC TTC TGC AAT TGA

[0303] were designed based on the available C. albicans PGK1 sequence(Genbank Accession No. U25180) were used to amplify a 700 bp fragmentfrom the region upstream of the C. albicans PGK1 open reading frame,using C. albicans genomic DNA as the template. Restriction sites XbaIand SpeI (underlined above) were added to the primers to facilitatecloning of the fragment. After amplification, the fragment was isolatedand digested with restriction enzymes XhoI and NcoI and then ligated toplasmid pMI203 digested with XhoI and NcoI. The resulting plasmid,pMI205, is shown in FIG. 16.

[0304] Isolation of C. sonorensis Genes

[0305] In order to develop appropriate genetic tools for C. sonorensis,a genomic library was constructed from this species. Genes of interestwere isolated from the library on the basis of known amino acid andnucleotide sequences of genes from related yeasts. Promoters andterminators of strong constitutively expressed genes such as PGK1 andTDH1 were isolated and used to express heterologous genes. The PDC geneswere isolated because the corresponding enzymes carry out reactions thatcompete with lactic acid production. Therefore it is desirable to deletethe PDC genes from the strains that will be used for lactic acidproduction.

[0306] Genomic DNA of C. sonorensis (ATCC Accession No. 32109) wasisolated from cells grown overnight in YPD using the Easy DNA kit(Invitrogen). DNA was partially digested with Sau3A and sizefractionated by sucrose gradient centrifugation (Sambrook et al. 1989,Molecular Cloning, 2^(nd) ed., Cold Spring Harbor Laboratory, N.Y.), andDNA fragments of about 22 kb were ligated to BamHI digested, phosphatasetreated lambda DASH™ vector arms (Stratagene) and the ligation mixturewas packaged into lambda particles using Gigapack II Gold PackagingExtract (Stratagene). The lambda particles were used to infect E. coliMRA P2.

[0307] The probes used for isolation of C. sonorensis genes from thelibrary were prepared by PCR amplification using the Dynazyme EXTpolymerase (Finnzymes, Espoo, Finland), sequence specific primers andgenomic DNA of S. cerevisiae, C. albicans or C. sonorensis as a templateas follows:.

[0308] Oligonucleotides TGT CAT CAC TGC TCC ATC TT (SEQ ID No.48) andTTA AGC CTT GGC AAC ATA TT (SEQ ID No. 49) corresponding to the S.cerevisiae TDH1 gene were used to amplify a fragment of the TDH genefrom genomic S. cerevisiae DNA.

[0309] Oligonucleotides GCG ATC TCG AGG TCC TAG AAT ATG TAT ACT AAT TTGC (SEQ ID No. 50) and CGC GAA TTC CCA TGG TTA GTT TTT GTT GGA AAG AGCAAC (SEQ ID No.51) corresponding to the C. albicans PGK1 gene were usedto amplify a fragment of the PGK1 gene from genomic C. albicans DNA.

[0310] Oligonucleotides TGG ACT AGT AAA CCA ACA GGG ATT GCC TTA GT (SEQID No. 52) and CTA GTC TAG AGA TCA TTA CGC CAG CAT CCT AGG (SEQ ID No.53) corresponding to the C. sonorensis 26 S rRNA were used to amplify afragment of the 26S rDNA gene from C. sonorensis genomic DNA.

[0311] Oligonucleotides CCG GAA TTC GAT ATC TGG GCW GGK AAT GCC AAY GARTTR AAT GC (SEQ ID No. 54) and CGC GGA TTC AGG CCT CAG TAN GAR AAW GAACCN GTR TTR AAR TC (SEQ ID No.55) were designed based on portions ofpyruvate decarboxylase amino acid sequence WAGNANELNA (SEQ ID No. 56)and DFNTGSFSYS (SEQ ID No. 57), that are conserved between S. cerevisiaePDC 1, Pichia stipitis PDC 1 and PDC2, and incomplete sequences ofCandida albicans PDC1 and PDC3. These primers were used were used toamplify a fragment of the PDC gene(s) from C. sonorensis genomic DNA.PCR reaction with these primers produced two fragments of differentnucleotide sequence termed PDC 1 and PDC2.

[0312] Oligonucleotides TCTGTTMCCTACRTAAGA (SEQ ID No. 58) andGTYGGTGGTCACGAAGGTGC (SEQ ID No. 59) were designed based on conservedregions found in fungal alcohol dehydrogenase sequences. These primerswere used to amplify a fragment of the ADH gene(s) from C. sonorensisgenomic DNA. PCR reaction with these primers produced three fragments ofdifferent nucleotide sequences termed ADH1, ADH2, and ADH3.

[0313] The library was screened with PCR fragments produced as describedabove, and products were labeled with ³²P α-dCTP using the Random PrimedLabeling Kit (Boehringer Mannheim). Hybridization with the radioactiveprobes was performed by incubation overnight at 42° C. in a solutioncontaining 50% formamide, 5× Denhardt's, 5× SSPE, 0.1% SDS, 100 μg/mLherring sperm DNA, 1 μg/mL polyA DNA. For TDH1, PGK1, and PDC1 probes,filters were washed after hybridization at room temperature in asolution of 2× SSC for 5 min and repeated, followed by two 30 min washesin a solution of 1× SSC-0.1% SDS at 68° C. The post hybridization washesfor rDNA and PDC2 probes were performed twice for 5 min at roomtemperature in 2× SSC, followed by two 30 min. washes in 0.1× SSC-0.1%SDS at 68° C.

[0314] Positive plaques were isolated and purified according tomanufacturers instructions (Stratagene). Bacteriophages were purifiedusing conventional methods (Sambrook et al., ibid.), modified byeliminating DNAseI treatment and precipitating phage particles releasedfrom lysed host cells using PEG6000, which phage particles were thendissolved in SM buffer and extracted with chloroform, pelleted bycentrifugation at 25,000 rpm in Kontron TST41.14 rotor for 2 h, andagain dissolved in SM buffer. Lambda DNA was isolated by digesting thephage particles with proteinase K followed by phenol extraction andethanol precipitation.

[0315] The C. sonorensis genomic DNA inserts were partially sequencedusing sequence-specific primers. The nucleotide sequences and the aminoacid sequences deduced therefrom were compared against sequence databases in order to identify genes encoded in whole or part by the phageinsert by homology to known genes or proteins. The sequences obtainedhad significant similarity to fungal rDNA, phosphoglycerate kinases,glyceraldehyde-3-phosphate dehydrogenases, or pyruvate decarboxylasesdepending on the probe used for isolating each clone. The start and endpoints of the open reading frames encoding sequences of C. sonorensisPGK1, PDC1 and TDH1 were identified thereby.

[0316] Use of MEL5 Gene Selection for Selecting C. sonorensisTransformants

[0317] In order to develop a positive selection for C. sonorensistransformants, the S. cerevisiae MEL5 gene (Naumov et al., 1990, MGG224:119-128; Turakainen et al., 1994, Yeast 10: 1559-1568; GenbankAccession No. Z37511) was obtained as the 2160 bp EcoRI-SpeI fragmentfrom plasmid pMEL5-39 and ligated to pBluescript II KS(−) (Stratagene)digested with EcoRI and SpeI cut. The EcoRI site in the MEL5 gene islocated 510 bp upstream of the initiator ATG, and the SpeI site islocated 250 bp downstream of the stop codon of MEL5. The resultingplasmid was designated pMI233, and is shown in FIG. 16.

[0318] The 1500 bp PGK1 promoter of C. sonorensis was amplified withprimers GCG ATC TCG AGA AAG AAA CGA CCC ATC CAA GTG ATG (SEQ ID No. 60)and TGG ACT AGT ACA TGC ATG CGG TGA GAA AGT AGA AAG CAA ACA TTG TAT ATAGTC TTT TCT ATT ATT AG (SEQ ID No. 61) using DNA from the PGK1 lambdaclone isolated above as template. The 3′ primer (SEQ ID No. 61) cancreate a fusion between the C. sonorensis PGK1 promoter and S.cerevisiae MEL5, since it corresponds to nucleotides present in the PGK1promoter immediately upstream of the open reading frame and nucleotidescorresponding to the 5′ end of MEL5 open reading frame. The resultingamplified fragment was digested with restriction enzymes SphI and Xholand ligated to plasmid pMI233 digested with SphI and XhoI. The resultingconstruct in the plasmid contains C. sonorensis PGK1 promoter upstreamof and operatively linked to the MEL5 open reading frame, and isidentified as pMI234 in FIG. 16.

[0319] In a similar fashion, a 650 bp of the C. sonorensis TDH1 promoterwas amplified with primers GCG ATC TCG AGA AAA TGT TAT TAT AAC ACT ACA C(SEQ ID No. 62) and TGG ACT AGT ACA TGC ATG CGG TGA GAA AGT AGA AAG CAAACA TTT TGT TTG ATT TGT TTG TTT TGT TTT TGT TTG (SEQ ID No. 63) usingDNA from the TDH1 lambda clone isolated above as the template. The 3′primer (SEQ ID No. 63) can create a fusion between C. sonorensis TDH1promoter and S. cerevisiae MEL5, since it corresponds to nucleotidespresent in the TDH1 promoter immediately upstream of the open readingframe and nucleotides corresponding to the 5′ end of MEL5 open readingframe. The amplified fragment was cut with SphI and XhoI and ligated toplasmid pMI233 digested with SphI and XhoI. The resulting plasmid,identified as pMI23 8 in FIG. 16, contains C. sonorensis TDH1 promoterupstream of and operatively linked to the MEL5 open reading frame.

[0320] The MEL5 expression cassette was released from vector sequencesby restriction enzyme digestion with SpeI and XhoI, and 1 μg of thelinear DNA was transformed into C. sonorensis by electroporationaccording to the method of Backer et al. (1999, Yeast 15: 1609-1618).Cells were transformed after growth overnight in 50 mL of YPD,harvesting by centrifugation and after being resuspended in a solutionof 0.1 M LiAc/10 mM DTT/10 mM Tris-HCl and incubated at room temperaturefor 1 h. The cells were collected by centrifugation and washed with coldwater and IM sorbitol and resuspended in cold 1M sorbitol. DNA (1-3 μg)was added into 40 μL of cell suspension and the mixture was pipettedinto a 0.2 cm electroporation cuvette. The Bio-Rad Gene Pulser was usedat settings of 1.5 kV, 25 F, 200 Ω for electroporation. After theelectric pulse 1 mL of cold 1M sorbitol was added onto the cells. Thecells were then incubated at 30° C. without shaking for 1 h;alternatively, 2 mL YPD was added and the incubation was continued at30° C. for 2-3 h. The cells were plated onto appropriate agar plates forregeneration.

[0321] In additional alternatives, the cells were transformed accordingto the protocol of Gietz et al. (1992, Nucleic Acids Res. 20:1425).Transformants were grown on YPD agar plates (comprising 10 g/L yeastextract, 20 g/L peptone 20 g/l, 20 g/L glucose and 2% agar) supplementedwith the chromogenic substrate of α-galactosidase,5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-gal; ICNBiochemicals) at a concentration of 40 μg/mL. The plates were incubatedat 30° C. for 1-3 days and then transferred to 4° C. In the presence ofX-gal yeast colonies transformed with a functional MEL5 expressioncassette turned blue, whereas the untransformed colonies were white. Thetransformation frequency obtained in these experiments was 2-20transformants/μg DNA. Blue colonies were purified by restreaking themonto fresh indicator plates.

[0322] The transformants also acquired the ability to grow on melibioseas the sole carbon source. This indicates that melibiose was hydrolysedinto glucose and galactose by the MELS-encoded α-galactosidase that wassecreted into the medium.

[0323] A parallel strategy to screening the transformants on X-galcontaining medium is selecting them on a medium containing melibiose asthe sole carbon source. After transformation the cells are spread ontoagar plates containing 0.67% Yeast Nitrogen base (Difco) and 2%melibiose and the plates are incubated at 30° C. for 5-10 days. Underthese conditions the untransformed cells are unable to grow to a visiblecolony whereas the MEL5 transformants form colonies.

[0324]L. helveticus LDH Expression Cassette and Vector Containing theMEL5 Marker for C. sonorensis

[0325] Plasmid pMI205 was used to produce a plasmid containing the MEL5gene as a selectable marker and the LDH gene for enabling production oflactic acid in C. soronensis. In the resulting plasmid, the zeocinresistance gene in pMI205 was replaced by the L. helveticus LDH gene.

[0326] A 1329 bp NcoI-BamHI fragment of pVR1 containing the LDH gene andthe CYC1 terminator was ligated to the 3413 bp NcoI-BamHI fragment ofpMI205 bringing the L. helveticus LDH gene under control of the C.albicans PGK1 promoter; the resulting plasmid was named pMI214 and isshown in FIG. 16. In a second step the C. albicans PGK1 promoter wasreplaced by the C. sonorensis PGK1 promoter. The C. sonorensis PGK1promoter was isolated by amplification from an isolated lambda clone asdescribed above using primers GCG ATC TCG AGA AAG AAA CGA CCC ATC CAAGTG ATG (SEQ ID No. 64) and ACT TGG CCA TGG TAT ATA GTC TTT TCT ATT ATTAG (SEQ ID No. 65), and the PCR product was digested with XhoI and Neoland ligated into pMI214 digested with Xhol and NcoI. This plasmid wasdesignated pMI277 and is shown in FIG. 16.

[0327] The LDH expression cassette from pMI227 and MELS marker cassettefrom pMI234 were combined into the same vector by ligating a 3377 bpAvrII-NheI fragment of pMI227 with SpeI-digested pMI234. The resultingplasmid was designated pMI246 and is shown in FIG. 16.

[0328] The LDH expression cassette from pMI227 and the MEL5 markercassette from pMI238 were combined into the same vector by ligating a3377 bp AvrII-NheI fragment of pMI227 with SpeI-digested pMI238. Theresulting plasmid was designated pMI247 and is shown in FIG. 16.

[0329] Transformation of C. sonorensis for Lactic Acid Production

[0330] LDH-encoding vectors were introduced into C. sonorensis cellsusing transformation methods developed in the art for other yeastspecies. Before transformation, plasmids pMI246 and pMI247 werelinearized by restriction enzyme digestion BstBI, an enzyme that cutswithin the rDNA sequences, thus targeting integration into the rDNAlocus. Alternatively, the transforming plasmids were digested with ApaIand BamHI, followed by purification from agarose gel, thereby releasingthe marker and LDH cassettes from vector sequences and facilitatingrandom integration in the genome.

[0331]C. sonorensis was transformed with pMI246 or pMI247 by the lithiumacetate method (Gietz et al., 1992, Nucleic Acids Res. 20:1425) or byelectroporation as described above and transformants were screened andpurified based on the blue color formed on YPD plates supplemented withX-gal.

[0332] Alpha-galactosidase producing colonies were tested for theproduction of lactic acid. The transformants were grown in YPD liquidmedium overnight at 30° C. and aliquots of the culture medium werewithdrawn and analyzed for the presence of lactic acid enzymaticallyusing an L-lactic acid determination kit (Boehringer Mannheim). At least10 times more lactic acid was detected in culture supernatants of thetransformants than those of the host strain.

[0333] Transformants originating from transformation of C. sonorensiswith BstBI cut pMI246 were designated as 246-1 through 246-9.Transformants originating from transformation of C. sonorensis withBstBI cut pMI247 were designated as 247-1 through 247-4. Transformantsoriginating from transformation of C. sonorensis with ApaI-BamHI cutpMI246 were designated as 246-10 through 246-15. Transformantsoriginating from transformation of C. sonorensis with ApaI-BamHI cutpMI247 were designated as 247-5 through 247-10.

[0334] Production of L-lactic Acid in Rich Media by C. sonorensisHarboring the L. helveticus LDH Gene Integrated into the Genome

[0335]C. sonorensis cells and the transformants disclosed above (246-1,246-2, 246-3, 247-1, 247-2, 247-3 and 247-4) were cultivated in YPDmedia. Precultures were grown in YPD-medium to an OD₆₀₀ of 11-17, andthen resuspended in 50 mL of YPD to an OD₆₀₀ of 0.5 for the cultivationexperiments. At the outset of cultivation, yeast cells were cultured in250 mL Erlenmeyer flasks with 250 rpm shaking. After 4 hours cultivationyeast cells were moved into 100 mL Erlenmeyer flasks and additionalglucose (corresponding to a final concentration of 20 g/L) was added andcultivations continued with 40 rpm shaking. Samples were withdrawnduring cultivation, OD₆₀₀, measured, and cells harvested bycentrifugation and the growth media analyzed by HPLC for lactic acid andglucose (using the L-lactic acid UV method and glucose/GOD-Periodatemethod of Boehringer Mannheim).

[0336] After 24 hours of cultivation transformants produced 2.4-3.3 g/Llactic acid (equivalent to 11-63% yield) from glucose, whereas controlstrain produced 0.05 g/L lactic acid (a 0.1% yield).

[0337] This example demonstrated that overexpression of LDH in C.sonorensis cells enhanced L-lactic acid production on glucose-containingmedia.

[0338] Production of L-lactic Acid in Minimal Glucose Media by C.sonorensis Harboring the L. helveticus LDH Gene Integrated into theGenome

[0339]C. sonorensis cells and the transformants disclosed above (246-1,246-3, 247-2) were cultivated in were cultivated in YD medium (yeastnitrogen base without amino acids supplemented-with 2% glucose).Precultures were grown in YD medium to an OD₆₀₀ of 10-13, cellscollected by centrifugation, washed once with YD medium and thenresuspended in 50 mL of YD to an OD₆₀₀ of 0.4 for the cultivationexperiments. Yeast were cultivated in 100 mL Erlenmeyer flasks with 40rpm shaking (microaerobic conditions). Samples were withdrawn duringcultivation, OD₆₀₀ measured, and cells harvested by centrifugation andthe growth media analyzed by HPLC for lactic acid and glucose. HPLCanalyses were carried out with a Waters 510 HPLC pump, Waters 717+autosampler and Water System Interphase Module liquid chromatographycomplex with refractive index detector (Waters 410 Differentialrefractometer) and UV-detector (Waters 2487 dual λ UV detector). AnAminex HPX-87H Ion Exclusion Column (300 mm×7.8 mm, Bio-Rad) used wasequilibrated with 5 mM H₂SO₄ in water at 35° C. and samples were elutedwith 5 mM H₂SO₄ in water at a flow rate of 0.6 mL/min. Data acquisitionand control was done with Waters Millennium software.

[0340] After 70 hours cultivation the transformants produced 2.2-2.5 g/Llactic acid (equivalent to 10-13% yield) from glucose, whereas thecontrol strain did not produce detectable lactic acid.

[0341] This example demonstrated that C. sonorensis cells overexpressinga heterologous LDH gene was capable of producing lactic acid fromglucose.

[0342] Production of L-lactic Acid in Minimal Glucose Media underAnaerobic Conditions by C. sonorensis Cells Harboring the L. helveticusLDH Gene Integrated into the Genome

[0343]C. sonorensis cells and the transformant disclosed above (246-1)were cultivated in were cultivated in YD medium (yeast nitrogen basewithout amino acids supplemented with 2% glucose) in anaerobic shakeflasks. Precultures were grown in YD medium to an OD₆₀₀ of 22-24, cellscollected by centrifugation, washed once with YD medium and thenresuspended in 100 mL of YD to an OD₆₀₀ of 0.75 for the cultivationexperiments. Yeast were cultivated in 100 mL Erlenmeyer flasks equippedwith waterlocks with 40 rpm shaking (anaerobic conditions). Samples werewithdrawn during cultivation, OD₆₀₀ measured, and cells harvested bycentrifugation and the growth media analyzed by HPLC for lactic acid andglucose. HPLC analyses were carried out with a Waters 510 HPLC pump,Waters 717+ autosampler and Water System Interphase Module liquidchromatography complex with refractive index detector (Waters 410Differential refractometer) and UV-detector (Waters 2487 dual λ UVdetector). An Aminex HPX-87H Ion Exclusion Column (300 mm×7.8 mm,Bio-Rad) used was equilibrated with 5 mM H₂SO₄ in water at 35° C. andsamples were eluted with 5 mM H₂SO₄ in water at a flow rate of 0.6mL/min. Data acquisition and control was done with Waters Millenniumsoftware.

[0344] After 160 hours of cultivation the strain 246-1 produced 1.1 g/Llactic acid (equivalent to 16% yield) from glucose, whereas the controlstrain produced lactic acid 0.03 g/L (0.1% yield).

[0345] This example demonstrated that C. sonorensis cells overexpressinga heterologous LDH gene was capable of producing lactic acid fromglucose under anaerobic conditions.

[0346] Production of L-lactic Acid in Minimal Xylose Media by C.sonorensis Harboring the L. helveticus LDH Gene Integrated into theGenome

[0347]C. sonorensis cells and the transformants (246-1, 246-3, 247-2)described above were cultivated in YX-medium (yeast nitrogen basewithout amino acids and supplemented with 2% xylose). Precultures weregrown in YPD-medium to an OD₆₀₀ of 10-13, and thereafter the cells werecollected by centrifugation, washed once with YX-medium and resuspendedto an OD₆₀₀ of 0.75 in 50 mL of YX-medium for cultivation experiments.Yeast cultures were cultivated in 100 mL Erlenmeyer flasks with 40 rpmshaking. Samples were withdrawn during cultivation, OD₆₀₀ measured, andcells harvested by centrifugation and the growth media analyzed by HPLCfor lactic acid and xylose. The HPLC analyses were carried out with aWaters 510 HPLC pump, Waters 717+ autosampler and Water SystemInterphase Module liquid chromatography complex with refractive indexdetector (Waters 410 Differential refractometer) and UV-detector (Waters2487 dual λ UV detector). An Aminex HPX-87H Ion Exclusion Column (300mm×7.8 mm, Bio-Rad) was equilibrated with 5 mM H₂SO₄ in water at 35° C.and samples were eluted with 5 mM H₂SO₄ in water at a flow rate of 0.6mL/min. Data acquisition and control was done with Waters Millenniumsoftware. L-lactic acid was analyzed by the L-lactic acid UV method ofBoehringer Mannheim.

[0348] After 70 hours of cultivation the transformants produced 0.1 g/Llactic acid (equivalent to 9-17% yields) from xylose, whereas thecontrol strain produced lactic acid 0.003 g/L (0.2% yield).

[0349] This example demonstrated that C. sonorensis overexpressing aheterologous lactate dehydrogenase encoding gene was capable ofproducing lactic acid from xylose.

[0350] Production of L-lactic Acid in Minimal Xylose Media by C.sonorensis Harboring the L. helveticus LDH Gene Integrated into theGenome

[0351]C. sonorensis cells and the transformants (246-1, 246-3, 247-2)described above were cultivated in YX-medium (yeast nitrogen basewithout amino acids and supplemented with 2% xylose). Precultures weregrown in YPD-medium to an OD₆₀₀ of 12-18, and thereafter the cells werecollected by centrifugation, washed once with YX-medium and resuspendedto an OD₆₀₀ of 2.0 in 50 mL of YX-medium for cultivation experiments.Yeast cultures were cultivated in 100 mL Erlenmeyer flasks with 40 rpmshaking (microaerobic conditions). Samples were withdrawn duringcultivation, OD₆₀₀ measured, and cells harvested by centrifugation andthe growth media analyzed by HPLC for lactic acid and xylose. T h e HPLCanalyses were carried out with a Waters 510 HPLC pump, Waters 717+autosampler and Water System Interphase Module liquid chromatographycomplex w i t h refractive index detector (Waters 410 Differentialrefractometer) and UV-detector (Waters 2487 dual λ UV detector). AnAminex HPX-87H Ion Exclusion Column (300 mm×7.8 mm, Bio-Rad) wasequilibrated with 5 mM H₂SO₄ in water at 35° C. and samples were elutedwith 5 mM H₂SO₄ in water at a flow rate of 0.6 mL/min. Data acquisitionand control was done with Waters Millennium software. L-lactic acid wasanalyzed by the L-lactic acid UV method of Boehringer Mannheim.

[0352] After 165 hours of cultivation the transformants produced 0.2 g/Llactic acid (equivalent to 5-6% yield) from xylose, whereas the controlstrain did not produce detectable lactic acid.

[0353] This example demonstrated that C. sonorensis overexpressing aheterologous lactate dehydrogenase encoding gene was capable ofproducing lactic acid from xylose.

[0354] Production of L-lactic Acid in Minimal Arabinose Media by C.sonorensis Harboring the L. helveticus LDH Gene Integrated into theGenome

[0355]C. sonorensis cells and the transformants (246-1, 246-3, 247-2)were cultivated in YA-medium (yeast nitrogen base without amino acidsand supplemented with 2% L-arabinose). Precultures were grown inYPD-medium to an OD₆₀₀ of 12-18, and thereafter the cells were collectedby centrifugation, washed once with YA-medium and resuspended to anOD₆₀₀ of 2.0 in 50 mL of YA-medium for cultivation experiments. Yeastcultures were cultivated in 100 mL Erlenmeyer flasks with 40 rpm shaking(microaerobic conditions). Samples were withdrawn during cultivation,OD₆₀₀ measured, and cells harvested by centrifugation and the growthmedia analyzed by HPLC for lactic acid and arabinose. The HPLC analyseswere carried out with a Waters 510 HPLC pump, Waters 717+ autosamplerand Water System Interphase Module liquid chromatography complex withrefractive index detector (Waters 410 Differential refractometer) andUV-detector (Waters 2487 dual λ UV detector). An Aminex HPX-87H IonExclusion Column (300 mm×7,8 mm, Bio-Rad) used was equilibrated with 5mM H₂SO₄ in water at 35° C., and samples were eluted with 5 mM H₂SO₄ inwater at a flow rate of 0.6 mL/min. Data acquisition and control wereperformed with the Waters Millennium software.

[0356] After 165 hours of cultivation the transformants produced0.04-0.05 g/L lactic acid (equivalent to a 2-3% yield) from arabinose,whereas the control strain produced lactic acid 0.007 g/L (a 0.5%yield).

[0357] This example demonstrated that C. sonorensis overexpressing aheterologous lactate dehydrogenase encoding gene was capable ofproducing lactic acid from arabinose.

[0358] Production of L-lactic Acid in Minimal Melibiose Media by C.sonorensis Strain Harboring the L. helveticus LDH Gene Integrated intothe Genome

[0359]C. sonorensis cells and the transformants described above (246-1,246-10, 247-2, 247-5) were cultivated in YM medium (yeast nitrogen basewithout amino acids supplemented with 2% melibiose). Precultures weregrown in YPD medium to an OD₆₀₀ of 18-25, cells were collected bycentrifugation and washed once with YM medium and resuspended in 50 mLof YM medium to an OD₆₀₀ of 1.5 for cultivation experiments. Yeast werecultivated in 100 mL Erlenmeyer flasks with 40 rpm shaking (microaerobicconditions). Samples were withdrawn during cultivation, OD₆₀₀ measured,and cells harvested by centrifugation and the growth media analyzed byHPLC for lactic acid (by the L-lactic acid UV method of BoehringerMannheim).

[0360] After 165 hours of cultivation the transformants produced 0.8-2.6g/L lactic acid.

[0361] This example discloses that C. sonorensis cells overexpressing aheterologous LDH gene was capable of producing lactic acid frommelibiose.

OTHER EMBODIMENTS

[0362] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1 65 1 92 DNA artificial sequence multiple cloning site 1 cccaagcttgaattccccgg gggatccctg cagggtacca cgcgtagatc tactagtgcg 60 gccgcctcgagtctagaggg cccaagcttg gg 92 2 91 DNA artificial sequence multiplecloning site 2 ccaagcttgg gccctctaga ctcgaggcgg ccgcactagt agatctacgcgtggtaccct 60 gcagggatcc cccggggaat tcaagcttgg g 91 3 31 DNALactobacillus helveticus 3 ccgggatcca tggcaagaga ggaaaaacct c 31 4 32DNA Lactobacillus helveticus 4 ccaagatctt tattgacgaa ccttaacgcc ag 32 537 DNA Pediococcus acidilactici 5 ccgggatcca tgtctaatat tcaaaatcatcaaaaag 37 6 33 DNA Pediococcus acidilactici 6 ccaagatctt tatttgtcttgtttttcagc aag 33 7 82 DNA Kluyveromyces marxianus 7 taaacagtacaatcgcaaag aaaagctcca cacccaaacc aaataattgc aatgcaactt 60 cttttctttttttttctttt ct 82 8 79 DNA Kluyveromyces marxianus 8 ttataaaatcattaaaatcc aaaatcgtaa tttatctctt tatcctctcc ctctctacat 60 gccggtagaggtgtggtca 79 9 1736 DNA kanamycin resistance gene 9 gtacaacttgagcaagttgt cgatcagctc ctcaaattgg tcctctgtaa cggatgactc 60 aacttgcacattaacttgaa gctcagtcga ttgagtgaac ttgatcaggt tgtgcagctg 120 gtcagcagcatagggaaaca cggcttttcc taccaaactc aaggaattat caaactctgc 180 aacacttgcgtatgcaggta gcaagggaaa tgtcatactt gaagtcggac agtgagtgta 240 gtcttgagaaattctgaagc cgtattttta ttatcagtga gtcagtcatc aggagatcct 300 ctacgccggacgcatcgtgg ccgacctgca gggggggggg gggcgctgag gtctgcctcg 360 tgaagaaggtgttgctgact cataccaggc ctgaatcgcc ccatcatcca gccagaaagt 420 gagggagccacggttgatga gagctttgtt gtaggtggac cagttggtga ttttgaactt 480 ttgctttgccacggaacggt ctgcgttgtc gggaagatgc gtgatctgat ccttcaactc 540 agcaaaagttcgatttattc aacaaagccg ccgtcccgtc aagtcagcgt aatgctctgc 600 cagtgttacaaccaattaac caattctgat tagaaaaact catcgagcat caaatgaaac 660 tgcaatttattcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat 720 gaaggagaaaactcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg 780 attccgactcgtccaacatc aatacaacct ttaatttccc ctcgtcaaaa ataaggttat 840 caagtgagaaatcaccatga gtgacgactg aatccggtga gaatggcaaa agcttatgca 900 ttctttccagacttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc 960 aaccaaaccgttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt 1020 aaaaggacaattacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc 1080 aacaatattttcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg 1140 gatcgcagtggtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg 1200 aagaggcataaattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc 1260 aacgctacctttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg 1320 atagattgtcgcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc 1380 agcatccatgttggaattta atcgcggcct cgagcaagac gtttcccgtt gaatatggct 1440 cataacaccccttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat 1500 atttttatcttgtgcaatgt aacatcagag attttgagac acaacgtggc tttccccccc 1560 ccccctgcaggtcggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga 1620 catcaccgatggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt 1680 gggtatggtggcaggccccg tggccggggg actgttgggc gccatctcct tgcatg 1736 10 372 DNAKluyveromyces marxianus 10 ccggttcttt ctcttactct tacaagacca agaacattgtcgaattccac tccgactaca 60 tcaaggtcag aaacgccact ttcccaggtg tccaaatgaagttcgtcttg caaaagttgt 120 tgaccaaggt caaggatgct gctaagggtt acaagccagttccagttcct cacgctccaa 180 gagacaacaa gccagttgct gactctactc cattgaagcaagaatgggtc tggactcaag 240 tcggtaagtt cctacaagaa ggtgatgttg ttctaactgaaaccggtacc tccgctttcg 300 gtatcaacca aacccacttc ccaaatgaca cctacggtatctcccaagtc ttgtggggtt 360 ccattggttt ca 372 11 747 DNA Kluyveromycesthermotolerans 11 ttaccactgt cttcggtctg ccaggtgact tcaatctgcg tctgttggacgagatctacg 60 aggtcgaggg tatgagatgg gccggtaact gtaacgagtt gaacgcttcttacgctgccg 120 acgcttacgc cagaatcaag ggtatgtcct gtttgatcac caccttcggtgtcggtgagt 180 tgtccgcttt gaacggtatc gccggttctt acgctgagca cgtcggtgtcttgcacattg 240 tcggtgtccc atccgtctcc gcccaggcca agcagctatt gttgcaccacaccttgggta 300 acggtgactt cactgtcttc cacagaatgt ccgccaacat ctctgagaccactgctatga 360 tcactgatct agctaccgcc ccatctgaga tcgacagatg tatcagaaccacctacatta 420 gacagagacc tgtctacttg ggtttgccat ctaacttcgt tgaccagatggtcccagcct 480 ctctattgga caccccaatt gacttggcct tgaagccaaa cgaccagcaggctgaggagg 540 aggtcatctc tactttgttg gagatgatca aggacgctaa gaacccagtcatcttggctg 600 acgcttgcgc ttccagacac gatgtcaagg ctgagaccaa gaagttgattgacatcactc 660 agttcccatc tttcgttacc ccaatgggta agggttccat tgacgagaagcacccaagat 720 tcggtggtgt ctacgtcggt accttgt 747 12 1738 DNA kanalycinresistance gene fragment 12 gtacaacttg agcaagttgt cgatcagctc ctcaaattggtcctctgtaa cggatgactc 60 aacttgcaca ttaacttgaa gctcagtcga ttgagtgaacttgatcaggt tgtgcagctg 120 gtcagcagca tagggaaaca cggcttttcc taccaaactcaaggaattat caaactctgc 180 aacacttgcg tatgcaggta gcaagggaaa tgtcatacttgaagtcggac agtgagtgta 240 gtcttgagaa attctgaagc cgtattttta ttatcagtgagtcagtcatc aggagatcct 300 ctacgccgga cgcatcgtgg ccgacctgca gggggggggggggcgctgag gtctgcctcg 360 tgaagaaggt gttgctgact cataccaggc ctgaatcgccccatcatcca gccagaaagt 420 gagggagcca cggttgatga gagctttgtt gtaggtggaccagttggtga ttttgaactt 480 ttgctttgcc acggaacggt ctgcgttgtc gggaagatgcgtgatctgat ccttcaactc 540 agcaaaagtt cgatttattc aacaaagccg ccgtcccgtcaagtcagcgt aatgctctgc 600 cagtgttaca accaattaac caattctgat tagaaaaactcatcgagcat caaatgaaac 660 tgcaatttat tcatatcagg attatcaata ccatatttttgaaaaagccg tttctgtaat 720 gaaggagaaa actcaccgag gcagttccat aggatggcaagatcctggta tcggtctgcg 780 attccgactc gtccaacatc aatacaacct attaatttcccctcgtcaaa aataaggtta 840 tcaagtgaga aatcaccatg agtgacgact gaatccggtgagaatggcaa aagcttatgc 900 atttctttcc agacttgttc aacaggccag ccattacgctcgtcatcaaa atcactcgca 960 tcaaccaaac cgttattcat tcgtgattgc gcctgagcgagacgaaatac gcgatcgctg 1020 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggcgcaggaacac tgccagcgca 1080 tcaacaatat tttcacctga atcaggatat tcttctaatacctggaatgc tgttttcccg 1140 gggatcgcag tggtgagtaa ccatgcatca tcaggagtacggataaaatg cttgatggtc 1200 ggaagaggca taaattccgt cagccagttt agtctgaccatctcatctgt aacatcattg 1260 gcaacgctac ctttgccatg tttcagaaac aactctggcgcatcgggctt cccatacaat 1320 cgatagattg tcgcacctga ttgcccgaca ttatcgcgagcccatttata cccatataaa 1380 tcagcatcca tgttggaatt taatcgcggc ctcgagcaagacgtttcccg ttgaatatgg 1440 ctcataacac cccttgtatt actgtttatg taagcagacagttttattgt tcatgatgat 1500 atatttttat cttgtgcaat gtaacatcag agattttgagacacaacgtg gctttccccc 1560 ccccccctgc aggtcggcat caccggcgcc acaggtgcggttgctggcgc ctatatcgcc 1620 gacatcaccg atggggaaga tcgggctcgc cacttcgggctcatgagcgc ttgtttcggc 1680 gtgggtatgg tggcaggccc cgtggccggg ggactgttgggcgccatctc cttgcatg 1738 13 17 DNA artificial sequence degenerateamplification primer 13 gtbatyggyt chggtac 17 14 17 DNA artificialsequence degenerate amplification primer 14 swrtcdccrt gytcacc 17 15 22DNA artificial sequence amplification primer 15 gtacagttct ggatactgct cg22 16 18 DNA artificial sequence amplification primer 16 acaggcatcgatgctgtc 18 17 19 DNA Kluyveromyces thermotolerans 17 gtgatgtcggcgatatagg 19 18 21 DNA Kluyveromyces thermotolerans 18 ctacttggagccactatcga c 21 19 21 DNA Kluyveromyces thermotolerans 19 gatctcctgctaagctcttg c 21 20 20 DNA Kluyveromyces thermotolerans 20 gcagttttggatattcatgc 20 21 972 DNA Kluyveromyces thermotolerans 21 atgttccaagatacaaagtc tcaagcagta agaactgatg ccaaaacagt aaaagttgtg 60 gtagtgggagtgggaagtgt tgggtctgcc acagcgtata cgttgcttct cagcggcatc 120 gtttccgagattgtccttat cgacgtgaac aaagacaaag cagagggtga aagcatggac 180 ttaaaccacgcagcaccttc aaatacaagg tctcgagcgg gtgattatcc tgactgcgct 240 ggcgcggccattgttattgt cacatgtggg attaaccaaa aaaatggaca aacaaggatg 300 gatcttgctgcaaaaaatgc caacattatg ctggaaatca tccccaatgt tgccaaatat 360 gctcctgataccatcctgct tattgccacg aatcctgtcg atgttttgac ctatattagc 420 tataaggcgtcagggtttcc actaagcaga gttatcggct caggtacagt tctggatact 480 gctcgttttaaatacatcct cggagagcac ttcaagatct catcggacag catcgatgcc 540 tgtgtaattggagaacatgg tgattcgggt gtgcctgtct ggtctcttac caacatcgac 600 ggcatgaagctccgggatta ctgcgaaaaa gccaaccaca tatttgatca gaatgcgttc 660 catagaatctttgagcaaac gcgagacgct gcttacgata tcatcaagcg caaaggctat 720 acttcatatggaatcgcagc gggattactt cgcatagtaa aggcgatttt agaggataca 780 ggatccacacttacagtttc aaccgttggt gattattttg gggttgaaca aattgctata 840 agcgtccctaccaaactcaa taaaagtggg gctcatcaag tggctgaact ttcactcgat 900 gagaaggaaatagaattgat ggaaaaatca gctagtcaga tcaaatcagt gattgagcat 960 ctggagatca at972 22 323 PRT Kluyveromyces thermotolerans 22 Met Phe Gln Asp Thr LysSer Gln Ala Val Arg Thr Asp Ala Lys Thr 1 5 10 15 Val Lys Val Val ValVal Gly Val Gly Ser Val Gly Ser Ala Thr Ala 20 25 30 Tyr Thr Leu Leu LeuSer Gly Ile Val Ser Glu Ile Val Leu Ile Asp 35 40 45 Val Asn Lys Asp LysAla Glu Gly Glu Ser Met Asp Leu Asn His Ala 50 55 60 Ala Pro Ser Asn ThrArg Ser Arg Ala Gly Asp Tyr Pro Asp Cys Ala 65 70 75 80 Gly Ala Ala IleVal Ile Val Thr Cys Gly Ile Asn Gln Lys Asn Gly 85 90 95 Gln Thr Arg MetAsp Leu Ala Ala Lys Asn Ala Asn Ile Met Leu Glu 100 105 110 Ile Ile ProAsn Val Ala Lys Tyr Ala Pro Asp Thr Ile Leu Leu Ile 115 120 125 Ala ThrAsn Pro Val Asp Val Leu Thr Tyr Ile Ser Tyr Lys Ala Ser 130 135 140 GlyPhe Pro Leu Ser Arg Val Ile Gly Ser Gly Thr Val Leu Asp Thr 145 150 155160 Ala Arg Phe Lys Tyr Ile Leu Gly Glu His Phe Lys Ile Ser Ser Asp 165170 175 Ser Ile Asp Ala Cys Val Ile Gly Glu His Gly Asp Gly Val Pro Val180 185 190 Trp Ser Leu Thr Asn Ile Asp Gly Met Lys Leu Arg Asp Tyr CysGlu 195 200 205 Lys Ala Asn His Ile Phe Asp Gln Asn Ala Phe His Arg IlePhe Glu 210 215 220 Gln Thr Arg Asp Ala Ala Tyr Asp Ile Ile Lys Arg LysGly Tyr Thr 225 230 235 240 Ser Tyr Gly Ile Ala Ala Gly Leu Leu Arg IleVal Lys Ala Ile Leu 245 250 255 Glu Asp Thr Gly Ser Thr Leu Thr Val SerThr Val Gly Asp Tyr Phe 260 265 270 Gly Val Glu Gln Ile Ala Ile Ser ValPro Thr Lys Leu Asn Lys Ser 275 280 285 Gly Ala His Gln Val Ala Glu LeuSer Leu Asp Glu Lys Glu Ile Glu 290 295 300 Leu Met Glu Lys Ser Ala SerGln Ile Lys Ser Val Ile Glu His Leu 305 310 315 320 Glu Ile Asn 23 20DNA artificial sequence degenerate amplification primer 23 gtyggtgchggtgchgthgg 20 24 17 DNA artificial sequence degenerate amplificationprimer 24 swrtcdccrt gytcbcc 17 25 27 DNA artificial sequenceamplification primer 25 atccacaaca gcttacacgt tattgag 27 26 28 DNAartificial sequence amplification primer 26 gtttggttgc tggaagtggtgttgatag 28 27 27 DNA artificial sequence amplification primer 27aacattgaat agcttgctca ggttgtg 27 28 28 DNA artificial sequenceamplification primer 28 gataataaac gcgttgacat ttcagatg 28 29 939 DNATorulaspora pretoriensis CDS (1)..(939) 29 atg cat aga tgt gct aaa gtggcc atc gtc ggt gcc ggc caa gtt gga 48 Met His Arg Cys Ala Lys Val AlaIle Val Gly Ala Gly Gln Val Gly 1 5 10 15 tcc aca aca gct tac acg ttatta ttg agt agt ttg gtt gct gaa gtg 96 Ser Thr Thr Ala Tyr Thr Leu LeuLeu Ser Ser Leu Val Ala Glu Val 20 25 30 gtg ttg ata gat gtc gat aaa agaaag gtc gaa ggc caa ttt atg gat 144 Val Leu Ile Asp Val Asp Lys Arg LysVal Glu Gly Gln Phe Met Asp 35 40 45 ctg aac cac gcg gct cct tta acg aaggag tca cga ttc agt gct ggg 192 Leu Asn His Ala Ala Pro Leu Thr Lys GluSer Arg Phe Ser Ala Gly 50 55 60 gac tat gaa agt tgt gct gat gct gcg gttgta atc gta acg ggc ggg 240 Asp Tyr Glu Ser Cys Ala Asp Ala Ala Val ValIle Val Thr Gly Gly 65 70 75 80 gct aat cag aaa cct ggt caa act aga atggag cta gcc gag agg aac 288 Ala Asn Gln Lys Pro Gly Gln Thr Arg Met GluLeu Ala Glu Arg Asn 85 90 95 gtt aaa atc atg cag gaa gtg atc cct aag attgtg aaa tac gcc ccc 336 Val Lys Ile Met Gln Glu Val Ile Pro Lys Ile ValLys Tyr Ala Pro 100 105 110 aac gca att ttg ctg att gca aca aac cct gtcgat gta ctt acc tat 384 Asn Ala Ile Leu Leu Ile Ala Thr Asn Pro Val AspVal Leu Thr Tyr 115 120 125 gct agt ttg aaa gcg tcg gga ttc cca gca agccgg gtt att ggt tct 432 Ala Ser Leu Lys Ala Ser Gly Phe Pro Ala Ser ArgVal Ile Gly Ser 130 135 140 ggg aca gtt ctc gac tct gct cgt ata cag cacaac ctg agc aag cta 480 Gly Thr Val Leu Asp Ser Ala Arg Ile Gln His AsnLeu Ser Lys Leu 145 150 155 160 ttc aat gtt tca tct gaa agt gtc aac gcgttt att atc ggg gaa cat 528 Phe Asn Val Ser Ser Glu Ser Val Asn Ala PheIle Ile Gly Glu His 165 170 175 ggt gac tca agt gtg ccc gtc tgg tcg cttgct gag att gcc ggc atg 576 Gly Asp Ser Ser Val Pro Val Trp Ser Leu AlaGlu Ile Ala Gly Met 180 185 190 aaa gtg gag gat tac tgt agg cag tcc aagaga aag ttt gac ccc agc 624 Lys Val Glu Asp Tyr Cys Arg Gln Ser Lys ArgLys Phe Asp Pro Ser 195 200 205 att ctg acc aaa ata tat gag gag tcg cgtgac gcg gca gcc tac atc 672 Ile Leu Thr Lys Ile Tyr Glu Glu Ser Arg AspAla Ala Ala Tyr Ile 210 215 220 ata gaa cgc aaa ggc tat acc aat ttc gggatt gca gca ggt ttg gct 720 Ile Glu Arg Lys Gly Tyr Thr Asn Phe Gly IleAla Ala Gly Leu Ala 225 230 235 240 agg ata gtg aga gct att ctg aga gatgaa ggt gcc cta tta act gtg 768 Arg Ile Val Arg Ala Ile Leu Arg Asp GluGly Ala Leu Leu Thr Val 245 250 255 tct act gta ggt gag cac ttt ggc atgaaa gat gtt tca ttg agt gtt 816 Ser Thr Val Gly Glu His Phe Gly Met LysAsp Val Ser Leu Ser Val 260 265 270 cca act agg gta gac agg agc ggc gctcac cat gtc gtc gac ctt ctg 864 Pro Thr Arg Val Asp Arg Ser Gly Ala HisHis Val Val Asp Leu Leu 275 280 285 cta aac gac aag gag ctg gag caa attaaa aca tct gga gcc aag ata 912 Leu Asn Asp Lys Glu Leu Glu Gln Ile LysThr Ser Gly Ala Lys Ile 290 295 300 aag tca gcc tgt gat gaa ctt ggc att939 Lys Ser Ala Cys Asp Glu Leu Gly Ile 305 310 30 313 PRT Torulasporapretoriensis 30 Met His Arg Cys Ala Lys Val Ala Ile Val Gly Ala Gly GlnVal Gly 1 5 10 15 Ser Thr Thr Ala Tyr Thr Leu Leu Leu Ser Ser Leu ValAla Glu Val 20 25 30 Val Leu Ile Asp Val Asp Lys Arg Lys Val Glu Gly GlnPhe Met Asp 35 40 45 Leu Asn His Ala Ala Pro Leu Thr Lys Glu Ser Arg PheSer Ala Gly 50 55 60 Asp Tyr Glu Ser Cys Ala Asp Ala Ala Val Val Ile ValThr Gly Gly 65 70 75 80 Ala Asn Gln Lys Pro Gly Gln Thr Arg Met Glu LeuAla Glu Arg Asn 85 90 95 Val Lys Ile Met Gln Glu Val Ile Pro Lys Ile ValLys Tyr Ala Pro 100 105 110 Asn Ala Ile Leu Leu Ile Ala Thr Asn Pro ValAsp Val Leu Thr Tyr 115 120 125 Ala Ser Leu Lys Ala Ser Gly Phe Pro AlaSer Arg Val Ile Gly Ser 130 135 140 Gly Thr Val Leu Asp Ser Ala Arg IleGln His Asn Leu Ser Lys Leu 145 150 155 160 Phe Asn Val Ser Ser Glu SerVal Asn Ala Phe Ile Ile Gly Glu His 165 170 175 Gly Asp Ser Ser Val ProVal Trp Ser Leu Ala Glu Ile Ala Gly Met 180 185 190 Lys Val Glu Asp TyrCys Arg Gln Ser Lys Arg Lys Phe Asp Pro Ser 195 200 205 Ile Leu Thr LysIle Tyr Glu Glu Ser Arg Asp Ala Ala Ala Tyr Ile 210 215 220 Ile Glu ArgLys Gly Tyr Thr Asn Phe Gly Ile Ala Ala Gly Leu Ala 225 230 235 240 ArgIle Val Arg Ala Ile Leu Arg Asp Glu Gly Ala Leu Leu Thr Val 245 250 255Ser Thr Val Gly Glu His Phe Gly Met Lys Asp Val Ser Leu Ser Val 260 265270 Pro Thr Arg Val Asp Arg Ser Gly Ala His His Val Val Asp Leu Leu 275280 285 Leu Asn Asp Lys Glu Leu Glu Gln Ile Lys Thr Ser Gly Ala Lys Ile290 295 300 Lys Ser Ala Cys Asp Glu Leu Gly Ile 305 310 31 21 DNABacillus megaterium 31 cctgagtcca cgtcattatt c 21 32 22 DNA Bacillusmegaterium 32 tgaagctatt tattcttgtt ac 22 33 27 DNA Bacillus megaterium33 gctctagatg aaaacacaat ttacacc 27 34 28 DNA Bacillus megaterium 34atggatcctt acacaaaagc tctgtcgc 28 35 26 DNA Rhizopus oryzae 35ctttattttt ctttacaata taattc 26 36 19 DNA Rhizopus oryzae 36 actagcagtgcaaaacatg 19 37 29 DNA Rhizopus oryzae 37 gctctagatg gtattacactcaaaggtcg 29 38 30 DNA Rhizopus oryzae 38 gctctagatc aacagctacttttagaaaag 30 39 28 DNA artificial sequence cloning site sequence 39aaatctagat gagccatatt caacggga 28 40 29 DNA artificial sequence cloningsite sequence 40 ccggatcctt agaaaaactc atcgagcat 29 41 36 DNAKluyveromyces thermotolerans 41 gctctagaat tatgttccaa gatacaaagt ctcaag36 42 34 DNA Kluyveromyces thermotolerans 42 ccggaattca tcctcaattgatctccagat gctc 34 43 2229 DNA Kluyveromyces thermotolerans 43gcggccgcgg atcgctcttc cgctatcgat taattttttt ttctttcctc tttttattaa 60ccttaatttt tattttagat tcctgacctt caactcaaga cgcacagata ttataacatc 120tgcacaatag gcatttgcaa gaattactcg tgagtaagga aagagtgagg aactatcgca 180tacctgcatt taaagatgcc gatttgggcg cgaatccttt attttggctt caccctcata 240ctattatcag ggccagaaaa aggaagtgtt tccctccttc ttgaattgat gttaccctca 300taaagcacgt ggcctcttat cgagaaagaa attaccgtcg ctcgtgattt gtttgcaaaa 360agaacaaaac tgaaaaaacc cagacacgct cgacttcctg tcttcctatt gattgcagct 420tccaatttcg tcacacaaca aggtcctagc gacggctcac aggttttgta acaagcaatc 480gaaggttctg gaatggcggg aaagggttta gtaccacatg ctatgatgcc cactgtgatc 540tccagagcaa agttcgttcg atcgtactgt tactctctct ctttcaaaca gaattgtccg 600aatcgtgtga caacaacagc ctgttctcac acactctttt cttctaacca agggggtggt 660ttagtttagt agaacctcgt gaaacttaca tttacatata tataaacttg cataaattgg 720tcaatgcaag aaatacatat ttggtctttt ctaattcgta gtttttcaag ttcttagatg 780ctttcttttt ctctttttta cagatcatca aggaagtaat tatctacttt ttacaacaaa 840tctagaatta tgttccaaga tacaaagtct caagcagtaa gaactgatgc caaaacagta 900aaagttgtgg tagtgggagt gggaagtgtt gggtctgcca cagcgtatac gttgcttctc 960agcggcatcg tttccgagat tgtccttatc gacgtgaaca aagacaaagc agagggtgaa 1020agcatggact taaaccacgc agcaccttca aatacaaggt ctcgagcggg tgattatcct 1080gactgcgctg gcgcggccat tgttattgtc acatgtggga ttaaccaaaa aaatggacaa 1140acaaggatgg atcttgctgc aaaaaatgcc aacattatgc tggaaatcat ccccaatgtt 1200gccaaatatg ctcctgatac catcctgctt attgccacga atcctgtcga tgttttgacc 1260tatattagct ataaggcgtc agggtttcca ctaagcagag ttatcggctc aggtacagtt 1320ctggatactg ctcgttttaa atacatcctc ggagagcact tcaagatctc atcggacagc 1380atcgatgcct gtgtaattgg agaacatggt gattcgggtg tgcctgtctg gtctcttacc 1440aacatcgacg gcatgaagct ccgggattac tgcgaaaaag ccaaccacat atttgatcag 1500aatgcgttcc atagaatctt tgagcaaacg cgagacgctg cttacgatat catcaagcgc 1560aaaggctata cttcatatgg aatcgcagcg ggattacttc gcatagtaaa ggcgatttta 1620gaggatacag gatccacact tacagtttca accgttggtg attattttgg ggttgaacaa 1680attgctataa gcgtccctac caaactcaat aaaagtgggg ctcatcaagt ggctgaactt 1740tcactcgatg agaaggaaat agaattgatg gaaaaatcag ctagtcagat caaatcagtg 1800attgagcatc tggagatcaa ttgaggatga attcggatcc ggtagataca ttgatgctat 1860caatccagag aactggaaag attgtgtagc cttgaaaaac ggtgaaactt acgggtccaa 1920gattgtctac agattttcct gatttgccag cttactatcc ttcttgaaaa tatgcactct 1980atatctttta gttcttaatt gcaacacata gatttgctgt ataacgaatt ttatgctatt 2040ttttaaattt ggagttcagt gataaaagtg tcacagcgaa tttcctcaca tgtagggacc 2100gaattgttta caagttctct gtaccaccat ggagacatca aaaattgaaa atctatggaa 2160agatatggac ggtagcaaca agaatatagc acgagccgcg gatttatttc gttacgcatg 2220cgcggccgc 2229 44 32 DNA Candida sonorensis 44 tggactagta aaccaacagggattgcctta gt 32 45 33 DNA Candida sonorensis 45 ctagtctaga gatcattacgccagcatcct agg 33 46 37 DNA Candida albicans 46 gcgatctcga ggtcctagaatatgtatact aatttgc 37 47 36 DNA Candida albicans 47 acttggccatggtgatagtt attcttctgc aattga 36 48 20 DNA Saccharomyces cerevisiae 48tgtcatcact gctccatctt 20 49 20 DNA Saccharomyces cerevisiae 49ttaagccttg gcaacatatt 20 50 37 DNA Candida albicans 50 gcgatctcgaggtcctagaa tatgtatact aatttgc 37 51 39 DNA Candida albicans 51cgcgaattcc catggttagt ttttgttgga aagagcaac 39 52 32 DNA Candidasonorensis 52 tggactagta aaccaacagg gattgcctta gt 32 53 33 DNA Candidasonorensis 53 ctagtctaga gatcattacg ccagcatcct agg 33 54 44 DNA Candidasonorensis 54 ccggaattcg atatctgggc wggkaatgcc aaygarttra atgc 44 55 44DNA Candida sonorensis misc_feature (33)..(33) primer that does notencode amino acid 55 cgcggattca ggcctcagta ngaraawgaa ccngtrttra artc 4456 10 PRT Candida sonorensis 56 Trp Ala Gly Asn Ala Asn Glu Leu Asn Ala1 5 10 57 10 PRT Candida sonorensis 57 Asp Phe Asn Thr Gly Ser Phe SerTyr Ser 1 5 10 58 18 DNA Candida sonorensis 58 tctgttmcct acrtaaga 18 5920 DNA Candida sonorensis 59 gtyggtggtc acgaaggtgc 20 60 36 DNA Candidasonorensis 60 gcgatctcga gaaagaaacg acccatccaa gtgatg 36 61 68 DNACandida sonorensis 61 tggactagta catgcatgcg gtgagaaagt agaaagcaaacattgtatat agtcttttct 60 attattag 68 62 34 DNA Candida sonorensis 62gcgatctcga gaaaatgtta ttataacact acac 34 63 75 DNA Candida sonorensis 63tggactagta catgcatgcg gtgagaaagt agaaagcaaa cattttgttt gatttgtttg 60ttttgttttt gtttg 75 64 36 DNA Candida sonorensis 64 gcgatctcgagaaagaaacg acccatccaa gtgatg 36 65 35 DNA Candida sonorensis 65acttggccat ggtatatagt cttttctatt attag 35

We claim:
 1. An isolated nucleic acid encoding a yeast lactate dehydrogenate protein having an amino acid sequence identified by Seq. ID No.
 22. 2. An isolated nucleic acid encoding a yeast lactate dehydrogenate protein having an amino acid sequence identified by Seq. ID No.
 30. 3. An isolated nucleic acid according to claim 1 encoding a yeast lactate dehydrogenate protein that hybridizes to a nucleic acid probe identified by Seq. ID No. 21 under high stringency conditions.
 4. An isolated nucleic acid according to claim 3 wherein hybridization is detected after washing under high stringency conditions.
 5. An isolated nucleic acid according to claim 2 encoding a yeast lactate dehydrogenate protein that hybridizes to a nucleic acid probe identified by Seq. ID No. 29 under high stringency conditions.
 6. An isolated nucleic acid according to claim 5 wherein hybridization is detected after washing under high stringency conditions.
 7. A recombinant expression construct comprising a nucleic acid having a nucleotide sequence encoding a yeast lactate dehydrogenate protein according to claims 1 or 2, wherein the nucleic acid is expressed in a yeast cell.
 8. A recombinant expression construct according to claim 7, further comprising a yeast promoter operably linked to the nucleic acid encoding a yeast lactate dehydrogenate protein.
 9. A recombinant expression construct according to claim 7, further comprising a yeast transcriptional terminator element operably linked to the nucleic acid encoding a yeast lactate dehydrogenate protein.
 10. A recombinant expression construct according to claim 7, further comprising a yeast replication element derived from a yeast 2-micron circle plasmid.
 11. A yeast cell transformed with the recombinant expression construct of claims 7, wherein the transformed cell expresses the yeast lactate dehydrogenate protein.
 12. A yeast cell according to claim 11, wherein the yeast cell is a yeast from genera Saccharomyces, Kluyveromyces, Hansenula, Candida, Trichosporon, Yamadazyma, Torulaspora or Pichia.
 13. A yeast cell according to claim 11, wherein the yeast cell expresses a crabtree-negative phenotype.
 14. A yeast cell according to claim 11, wherein the yeast cell is a yeast species selected from the group consisting of C. soronensis and K. marxianus.
 15. A yeast cell according to claim 11, wherein the yeast cell produces a reduced amount of a glycolytic enzyme selected from the group consisting of pyruvate decarboxylase, alcohol dehydrogenate, and acetyl-CoA synthase.
 16. A method for producing lactic acid comprising the step of fermenting a yeast cell culture according to claim 11 in a nutrient medium containing a sugar under conditions whereby at least 50% of the sugar is converted by the yeast cell to lactic acid.
 17. The method of claim 16, wherein the yeast cell is grown at a temperature from about 35° C. to about 55° C.
 18. The method of claim 16, wherein the nutrient culture has a pH less than about pH 5.0.
 19. The method of claim 16, wherein the yeast is grown under substantially anaerobic conditions.
 20. The method of claim 16, wherein the yeast cell is a crabtree-negative yeast cell
 21. The method of claim 20, wherein the yeast cell is K. marxianus or C. sonorensis.
 22. The method of claim 16, wherein the yeast cell produces a reduced amount of a glycolytic enzyme selected from the group consisting of pyruvate decarboxylase, alcohol dehydrogenate, and acetyl-CoA synthase.
 23. The method of claim 16, wherein the sugar is glucose, xylose, ribose, arabinose, mannose, galactose, fructose, maltose or lyxose. 